1 Libraries and environment

1.1 Load environment

Libraries used to create and generate this report:

  • R : R version 4.2.1 (2022-06-23)
  • rmarkdown : 2.22
  • knitr : 1.43
  • rmdformats : 1.0.4
  • bookdown : 0.34
  • kableExtra : 1.3.4

1.2 Load libraries

Libraries used for data analysis:

library("DESeq2")
library("limma")
library("FactoMineR")
library("factoextra")
library("ggplot2")
library("pheatmap")
library("rRoma")
library("clusterProfiler")
library("org.Hs.eg.db")
library("ggrepel")
library("dendextend")
  • DESeq2: 1.38.3
  • limma: 3.54.2
  • FactoMineR: 2.8
  • ggplot2: 3.4.2
  • pheatmap: 1.0.12
  • clusterProfiler: 4.6.2
  • org.Hs.eg.db: 3.16.0
  • rRoma: 0.0.4.2000

2 Description of the RNA-seq dataset

In this tutorial, we will try a typical workflow for RNA-sequencing data analysis. We will use the TB dataset, which consists of gene expression profiles on 41 tumor biopsies from cancer patients.

  • RNA-seq is the high-troughput sequencing of cDNA using NGS technologies
  • RNA-seq works by sequencing evry RNA molecule and profiling the expression of genes by counting the number of times their transcripts have been sequenced.
  • The summarized RNA-seq output is a count matrix.

You can read more about how the data was processed here. In summary, it follows a general outline as in the figure below: first, we have a set of reads. Using an aligner and a reference genome, we determine the origin of these reads in terms of the chromosome and position within the reference genome. Then, we compare this information with a reference annotation that provides the chromosome and position details of exons in known genes.

3 Input data

In this matrix, each row represents a gene, each column a patient sample and the values provide the raw gene expression count numbers.

rna <- read.csv("data_tb/data_rna_count.csv", header = TRUE, row.names = 1)

dim(rna)
## [1] 19690    41

We have some information about the sample in the sample_annotation file. We downlaod prior knowledge of experimental design for patient samples.

design <- read.csv("data_tb/design.csv", header = TRUE, row.names = 1)
design <- design[colnames(rna),]
hospital <- as.factor(design$Hospital)
group <- as.factor(design$Group)
design$Hospital <- as.factor(design$Hospital)
design$Group <- as.factor(design$Group)

Tumor biopsies are collected from different hospitals and assigned to a unique sample ID. Clinical classification of these tumors based primarily on morphology (histopathology) and available clinicopathological parameters identified four different disease subgroups that are associated to different outcomes and are used to direct treatment strategies.

With recent developments in omics profiling, there have been significant advancements in the field of tumor classification, leading to a deeper understanding of the molecular characteristics and heterogeneity of tumors. This enhanced classification allows for more precise and personalized treatment strategies, as it helps to identify predictive biomarkers and potential therapeutic targets specific to each tumor subgroup.

4 Quality control of raw counts

Our count matrix contains many rows with only zeros, and additionally many rows with only a few count total. In order to reduce the size of the matrix, and to increase the speed of our analyses, we can remove the rows that have no or nearly no information about the amount of gene expression.

g.zero <- rowSums(rna == 0) # Number of zero counts per gene
s.zero <- colSums(rna == 0) # Number of features (genes) with zero counts per sample

barplot(colSums(rna), main = "Total counts per sample", ylab = "Total counts", las=1, las=2, cex.axis= 0.8, cex.names=0.8 )
grid()

barplot(s.zero/nrow(rna), main = "Proportion of null features per sample", ylab = "Proportion of null counts", las=2, cex.names=0.8 )
grid()

dim(rna)
## [1] 19690    41
keep <- rowSums(rna) >= 10 # Keep only rows that have at least 10 reads total.
rna <- rna[keep,]

dim(rna)
## [1] 18241    41

5 Normalization

Box plot for the gene expression data before normalization. The black bar indicates the median value. These distributions need to be similar for the different samples to be comparable. If this is not the case, the data should be normalised.

boxplot(log(rna + 1))

We create a DESeq object from the data matrix and apply normalization by DESeq method

dds <- DESeqDataSetFromMatrix(countData = rna, colData = design, design = ~ Hospital+Group) 
### DESeq normalization
dds <- estimateSizeFactors(dds)
dds <- estimateDispersions(dds)

rna.norm <- counts(dds, normalized=TRUE)
boxplot(log(rna.norm+1))

log.rna.norm <- log(rna.norm+1)

6 Principal component analysis

We can use principal component analysis (PCA) for reducing our large scale data matrix into few major components. Exploratory analysis by PCA is crucial for quality control and gaining initial insights into our dataset. It can help us detect quality problems, sample swaps and contamination, as well as give us a sense of the most salient patterns present in the data, their structure and variability.

We use FactoMineR, an R package dedicated to multidimensional exploratory analysis of data.

pca.rna <- PCA(t(log.rna.norm), graph = FALSE)

6.1 Explore PCA results

The proportion of variances retained by the principal components can be visualized by with a scree plot, which is a graph of the eigenvalues/variances associated with components.

## display eigen values
fviz_eig(pca.rna)

Other functions are available to visualize samples in the PCA space:

## display samples
fviz_pca_ind(pca.rna, habillage = design$Group)

fviz_pca_ind(pca.rna, habillage = design$Group, axes = c(3,4))

Question: how many tumor subgroups do you observe?

We can further explore which of the original variables are contributing the most to the various PCs by inspecting their barplot.

## most contributing genes 
fviz_contrib(pca.rna, choice = "var", top = 30, axes = 1)

fviz_contrib(pca.rna, choice = "var", top = 30, axes = 2)

7 Clustering

Unsupervised clustering methods are widely used for data exploration and generating hypotheses. The aim is to partition data according to natural classes present in it, assigning samples that are “more similar” to the same “cluster”.

To perform clustering, the first step is to compute the distance between each sample:

d <- dist(t(log.rna.norm))

We can perform hierarchical clustering based on the distances defined above using the hclust function. This function returns an hclust object that describes the groupings that were created using the algorithm described above. The plot method represents these relationships with a tree or dendrogram:

hc <- hclust(d, method = "ward.D")
plot(hc, hang = -1)
dend <- as.dendrogram(hc)
colored_bars(as.numeric(design$Group), dend = dend)

Based on the dendogram, we can cut the tree into k clusters that represents our classes.

8 Differential expression for RNA-seq

The standard differential expression analysis steps are performed by a single function, DESeq. Results tables are generated using the function results, which extracts a results table with log2 fold changes, p values and adjusted p values. We can select significantly different expressed genes according to a given p-value threshold

dds <- DESeq(dds)
res <- results(dds, contrast = c("Group", "G4", "G3"))
sum(res$padj<=0.05, na.rm = TRUE)
## [1] 4065

8.1 Heatmap visualization

We can visualize our results with a heatmap. Here we display the top 50 most differentially expressed genes.

res <- res[order(res$padj),]
top50 <- res[1:50, ]
top50.mat <- log.rna.norm[rownames(top50),]
pheatmap(top50.mat, scale="row", annotation_col = design, clustering_distance_rows = "correlation", clustering_method = "ward.D")

9 Downstream analysis and gene-set testing

The volcano plot is a classical representation of the results of a differential expression analysis. In such a plot we display the -log10 of the pvalue as a function of the log2 fold change. Here we also display the names of the 10 genes with the lowest pvalues as an example.

res_df <- as.data.frame(res)
res_df$symbol <- rownames(res_df)

ggplot(as.data.frame(res), aes(x = log2FoldChange, y = -log10(pvalue))) + 
  geom_point() + 
  geom_label_repel(aes(label = symbol), data = res_df[1:10,]) + 
  theme_light()

To interpret the list of differentially expressed genes, we can look for common biological functions among them. Several databases exist to link genes and biological functions, one the most used is Gene Ontology (GO). The enrichGO function from clusterProfiler will search for gene sets that are enriched in GO categories.

## select list of differentially expressed genes with sufficient logFC
genes_signif <- rownames(res)[which(res$padj <= 0.05 & abs(res$log2FoldChange)>=1)]

res_GO <- enrichGO(gene = genes_signif, OrgDb = "org.Hs.eg.db", keyType = "SYMBOL", universe = rownames(res), ont = "BP")
as.data.frame(res_GO)
##                    ID
## GO:0002181 GO:0002181
## GO:0042255 GO:0042255
## GO:0007156 GO:0007156
## GO:0042273 GO:0042273
## GO:0098742 GO:0098742
## GO:0042274 GO:0042274
## GO:0000027 GO:0000027
## GO:0042254 GO:0042254
## GO:0034765 GO:0034765
## GO:0050804 GO:0050804
## GO:0099177 GO:0099177
## GO:0098657 GO:0098657
## GO:0061564 GO:0061564
## GO:0050808 GO:0050808
## GO:0006364 GO:0006364
## GO:0071805 GO:0071805
## GO:0097485 GO:0097485
## GO:0001764 GO:0001764
## GO:0006813 GO:0006813
## GO:0050878 GO:0050878
## GO:0016072 GO:0016072
## GO:0007411 GO:0007411
## GO:0007409 GO:0007409
## GO:0006836 GO:0006836
## GO:0043270 GO:0043270
## GO:0030900 GO:0030900
## GO:2000027 GO:2000027
## GO:0000028 GO:0000028
## GO:0042391 GO:0042391
## GO:1904062 GO:1904062
## GO:0048880 GO:0048880
## GO:0098659 GO:0098659
## GO:0099587 GO:0099587
## GO:0021953 GO:0021953
## GO:0007589 GO:0007589
## GO:0001505 GO:0001505
## GO:0150063 GO:0150063
## GO:1904667 GO:1904667
## GO:0098739 GO:0098739
## GO:0006936 GO:0006936
## GO:0001654 GO:0001654
## GO:0032412 GO:0032412
## GO:0006865 GO:0006865
## GO:0022613 GO:0022613
## GO:0022898 GO:0022898
## GO:0030198 GO:0030198
## GO:0098656 GO:0098656
## GO:0043062 GO:0043062
## GO:0015701 GO:0015701
## GO:0045229 GO:0045229
## GO:0010959 GO:0010959
## GO:0021537 GO:0021537
## GO:0030879 GO:0030879
## GO:0048568 GO:0048568
## GO:0023061 GO:0023061
## GO:1990573 GO:1990573
## GO:0070252 GO:0070252
## GO:0002027 GO:0002027
## GO:0048708 GO:0048708
## GO:0010975 GO:0010975
## GO:0043931 GO:0043931
## GO:0000462 GO:0000462
## GO:0007595 GO:0007595
## GO:0140694 GO:0140694
## GO:1901796 GO:1901796
## GO:0007193 GO:0007193
## GO:0015800 GO:0015800
## GO:0007157 GO:0007157
## GO:0051444 GO:0051444
## GO:0021534 GO:0021534
## GO:0021924 GO:0021924
## GO:0021930 GO:0021930
## GO:0070977 GO:0070977
## GO:0048799 GO:0048799
## GO:0042363 GO:0042363
## GO:1904816 GO:1904816
## GO:0042476 GO:0042476
## GO:0014068 GO:0014068
## GO:1904064 GO:1904064
## GO:0032409 GO:0032409
## GO:0086003 GO:0086003
## GO:0003012 GO:0003012
## GO:0086011 GO:0086011
## GO:0001504 GO:0001504
## GO:0030490 GO:0030490
## GO:0043266 GO:0043266
## GO:0015844 GO:0015844
##                                                                                         Description
## GO:0002181                                                                  cytoplasmic translation
## GO:0042255                                                                        ribosome assembly
## GO:0007156                          homophilic cell adhesion via plasma membrane adhesion molecules
## GO:0042273                                                       ribosomal large subunit biogenesis
## GO:0098742                                cell-cell adhesion via plasma-membrane adhesion molecules
## GO:0042274                                                       ribosomal small subunit biogenesis
## GO:0000027                                                         ribosomal large subunit assembly
## GO:0042254                                                                      ribosome biogenesis
## GO:0034765                                                regulation of ion transmembrane transport
## GO:0050804                                             modulation of chemical synaptic transmission
## GO:0099177                                                   regulation of trans-synaptic signaling
## GO:0098657                                                                         import into cell
## GO:0061564                                                                         axon development
## GO:0050808                                                                     synapse organization
## GO:0006364                                                                          rRNA processing
## GO:0071805                                                    potassium ion transmembrane transport
## GO:0097485                                                               neuron projection guidance
## GO:0001764                                                                         neuron migration
## GO:0006813                                                                  potassium ion transport
## GO:0050878                                                          regulation of body fluid levels
## GO:0016072                                                                   rRNA metabolic process
## GO:0007411                                                                            axon guidance
## GO:0007409                                                                             axonogenesis
## GO:0006836                                                               neurotransmitter transport
## GO:0043270                                                     positive regulation of ion transport
## GO:0030900                                                                    forebrain development
## GO:2000027                                                 regulation of animal organ morphogenesis
## GO:0000028                                                         ribosomal small subunit assembly
## GO:0042391                                                         regulation of membrane potential
## GO:1904062                                             regulation of cation transmembrane transport
## GO:0048880                                                               sensory system development
## GO:0098659                                           inorganic cation import across plasma membrane
## GO:0099587                                              inorganic ion import across plasma membrane
## GO:0021953                                            central nervous system neuron differentiation
## GO:0007589                                                                     body fluid secretion
## GO:0001505                                                    regulation of neurotransmitter levels
## GO:0150063                                                                visual system development
## GO:1904667                                 negative regulation of ubiquitin protein ligase activity
## GO:0098739                                                            import across plasma membrane
## GO:0006936                                                                       muscle contraction
## GO:0001654                                                                          eye development
## GO:0032412                                     regulation of ion transmembrane transporter activity
## GO:0006865                                                                     amino acid transport
## GO:0022613                                                     ribonucleoprotein complex biogenesis
## GO:0022898                                         regulation of transmembrane transporter activity
## GO:0030198                                                        extracellular matrix organization
## GO:0098656                                                            anion transmembrane transport
## GO:0043062                                                     extracellular structure organization
## GO:0015701                                                                    bicarbonate transport
## GO:0045229                                            external encapsulating structure organization
## GO:0010959                                                        regulation of metal ion transport
## GO:0021537                                                                telencephalon development
## GO:0030879                                                                mammary gland development
## GO:0048568                                                              embryonic organ development
## GO:0023061                                                                           signal release
## GO:1990573                                              potassium ion import across plasma membrane
## GO:0070252                                                          actin-mediated cell contraction
## GO:0002027                                                                 regulation of heart rate
## GO:0048708                                                                astrocyte differentiation
## GO:0010975                                              regulation of neuron projection development
## GO:0043931                                                 ossification involved in bone maturation
## GO:0000462 maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA)
## GO:0007595                                                                                lactation
## GO:0140694                                                  non-membrane-bounded organelle assembly
## GO:1901796                                  regulation of signal transduction by p53 class mediator
## GO:0007193                adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway
## GO:0015800                                                              acidic amino acid transport
## GO:0007157              heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules
## GO:0051444                            negative regulation of ubiquitin-protein transferase activity
## GO:0021534                                                          cell proliferation in hindbrain
## GO:0021924                                             cell proliferation in external granule layer
## GO:0021930                                          cerebellar granule cell precursor proliferation
## GO:0070977                                                                          bone maturation
## GO:0048799                                                                  animal organ maturation
## GO:0042363                                                    fat-soluble vitamin catabolic process
## GO:1904816              positive regulation of protein localization to chromosome, telomeric region
## GO:0042476                                                                            odontogenesis
## GO:0014068                           positive regulation of phosphatidylinositol 3-kinase signaling
## GO:1904064                                    positive regulation of cation transmembrane transport
## GO:0032409                                                       regulation of transporter activity
## GO:0086003                                                          cardiac muscle cell contraction
## GO:0003012                                                                    muscle system process
## GO:0086011                                          membrane repolarization during action potential
## GO:0001504                                                                  neurotransmitter uptake
## GO:0030490                                                                   maturation of SSU-rRNA
## GO:0043266                                                    regulation of potassium ion transport
## GO:0015844                                                                      monoamine transport
##            GeneRatio   BgRatio       pvalue     p.adjust       qvalue
## GO:0002181   98/2229 158/15817 2.538122e-44 1.505360e-40 1.405318e-40
## GO:0042255   28/2229  58/15817 4.922821e-10 1.459863e-06 1.362844e-06
## GO:0007156   53/2229 165/15817 2.471192e-09 4.885546e-06 4.560867e-06
## GO:0042273   31/2229  75/15817 6.820617e-09 1.011327e-05 9.441170e-06
## GO:0098742   72/2229 263/15817 1.022653e-08 1.213072e-05 1.132454e-05
## GO:0042274   29/2229  75/15817 1.200310e-07 1.121182e-04 1.046671e-04
## GO:0000027   15/2229  25/15817 1.323263e-07 1.121182e-04 1.046671e-04
## GO:0042254   75/2229 298/15817 2.271932e-07 1.684353e-04 1.572416e-04
## GO:0034765  109/2229 486/15817 3.221428e-07 2.122921e-04 1.981838e-04
## GO:0050804   96/2229 422/15817 8.319703e-07 4.934416e-04 4.606488e-04
## GO:0099177   96/2229 423/15817 9.303482e-07 5.016268e-04 4.682901e-04
## GO:0098657   61/2229 238/15817 1.561004e-06 7.715263e-04 7.202528e-04
## GO:0061564  104/2229 474/15817 1.763874e-06 8.047337e-04 7.512533e-04
## GO:0050808   93/2229 419/15817 3.720523e-06 1.576173e-03 1.471425e-03
## GO:0006364   56/2229 219/15817 4.445312e-06 1.757676e-03 1.640866e-03
## GO:0071805   53/2229 204/15817 4.818374e-06 1.786111e-03 1.667411e-03
## GO:0097485   58/2229 232/15817 6.428523e-06 2.242798e-03 2.093748e-03
## GO:0001764   47/2229 176/15817 7.393306e-06 2.402239e-03 2.242593e-03
## GO:0006813   57/2229 228/15817 7.695590e-06 2.402239e-03 2.242593e-03
## GO:0050878   81/2229 359/15817 8.168310e-06 2.422312e-03 2.261332e-03
## GO:0016072   62/2229 256/15817 9.341572e-06 2.638327e-03 2.462991e-03
## GO:0007411   57/2229 231/15817 1.170663e-05 3.156001e-03 2.946262e-03
## GO:0007409   92/2229 428/15817 1.628615e-05 4.199703e-03 3.920602e-03
## GO:0006836   52/2229 208/15817 1.892956e-05 4.531275e-03 4.230139e-03
## GO:0043270   65/2229 278/15817 1.927060e-05 4.531275e-03 4.230139e-03
## GO:0030900   82/2229 373/15817 1.986396e-05 4.531275e-03 4.230139e-03
## GO:2000027   36/2229 127/15817 2.082378e-05 4.574290e-03 4.270296e-03
## GO:0000028   10/2229  17/15817 2.278974e-05 4.827356e-03 4.506543e-03
## GO:0042391   88/2229 409/15817 2.378506e-05 4.864454e-03 4.541176e-03
## GO:1904062   80/2229 364/15817 2.520895e-05 4.953832e-03 4.624614e-03
## GO:0048880   84/2229 387/15817 2.589257e-05 4.953832e-03 4.624614e-03
## GO:0098659   33/2229 114/15817 2.838788e-05 5.102077e-03 4.763007e-03
## GO:0099587   33/2229 114/15817 2.838788e-05 5.102077e-03 4.763007e-03
## GO:0021953   44/2229 171/15817 3.836163e-05 6.691848e-03 6.247126e-03
## GO:0007589   25/2229  78/15817 4.077401e-05 6.909447e-03 6.450264e-03
## GO:0001505   52/2229 214/15817 4.321166e-05 6.983417e-03 6.519318e-03
## GO:0150063   82/2229 381/15817 4.403222e-05 6.983417e-03 6.519318e-03
## GO:1904667    8/2229  12/15817 4.474285e-05 6.983417e-03 6.519318e-03
## GO:0098739   47/2229 188/15817 4.663046e-05 7.091417e-03 6.620141e-03
## GO:0006936   73/2229 331/15817 4.974009e-05 7.375211e-03 6.885075e-03
## GO:0001654   81/2229 377/15817 5.202941e-05 7.526498e-03 7.026308e-03
## GO:0032412   60/2229 261/15817 6.652474e-05 9.394243e-03 8.769928e-03
## GO:0006865   37/2229 140/15817 8.419533e-05 1.161308e-02 1.084131e-02
## GO:0022613   91/2229 442/15817 9.751629e-05 1.296130e-02 1.209993e-02
## GO:0022898   61/2229 270/15817 9.834071e-05 1.296130e-02 1.209993e-02
## GO:0030198   66/2229 299/15817 1.087884e-04 1.402661e-02 1.309444e-02
## GO:0098656   58/2229 255/15817 1.188275e-04 1.490806e-02 1.391731e-02
## GO:0043062   66/2229 300/15817 1.206520e-04 1.490806e-02 1.391731e-02
## GO:0015701   12/2229  27/15817 1.317322e-04 1.594497e-02 1.488531e-02
## GO:0045229   66/2229 302/15817 1.479920e-04 1.755481e-02 1.638816e-02
## GO:0010959   81/2229 389/15817 1.568826e-04 1.824452e-02 1.703204e-02
## GO:0021537   56/2229 247/15817 1.686828e-04 1.923958e-02 1.796097e-02
## GO:0030879   34/2229 130/15817 1.989948e-04 2.226864e-02 2.078873e-02
## GO:0048568   88/2229 434/15817 2.175041e-04 2.374094e-02 2.216318e-02
## GO:0023061   90/2229 446/15817 2.201570e-04 2.374094e-02 2.216318e-02
## GO:1990573   17/2229  49/15817 2.333411e-04 2.471332e-02 2.307094e-02
## GO:0070252   27/2229  96/15817 2.472061e-04 2.551648e-02 2.382072e-02
## GO:0002027   28/2229 101/15817 2.495288e-04 2.551648e-02 2.382072e-02
## GO:0048708   23/2229  77/15817 2.670971e-04 2.685004e-02 2.506566e-02
## GO:0010975   87/2229 431/15817 2.751597e-04 2.719953e-02 2.539193e-02
## GO:0043931   11/2229  25/15817 2.803486e-04 2.722754e-02 2.541808e-02
## GO:0000462   14/2229  37/15817 2.934093e-04 2.722754e-02 2.541808e-02
## GO:0007595   14/2229  37/15817 2.934093e-04 2.722754e-02 2.541808e-02
## GO:0140694   77/2229 373/15817 2.977323e-04 2.722754e-02 2.541808e-02
## GO:1901796   28/2229 102/15817 2.983966e-04 2.722754e-02 2.541808e-02
## GO:0007193   22/2229  73/15817 3.132815e-04 2.815262e-02 2.628167e-02
## GO:0015800   20/2229  64/15817 3.407865e-04 3.016723e-02 2.816240e-02
## GO:0007157   15/2229  42/15817 3.754005e-04 3.223666e-02 3.009430e-02
## GO:0051444   10/2229  22/15817 3.860899e-04 3.223666e-02 3.009430e-02
## GO:0021534    8/2229  15/15817 3.913404e-04 3.223666e-02 3.009430e-02
## GO:0021924    8/2229  15/15817 3.913404e-04 3.223666e-02 3.009430e-02
## GO:0021930    8/2229  15/15817 3.913404e-04 3.223666e-02 3.009430e-02
## GO:0070977   11/2229  26/15817 4.244068e-04 3.448160e-02 3.219005e-02
## GO:0048799   12/2229  30/15817 4.353540e-04 3.489303e-02 3.257414e-02
## GO:0042363    7/2229  12/15817 4.513560e-04 3.511623e-02 3.278250e-02
## GO:1904816    7/2229  12/15817 4.513560e-04 3.511623e-02 3.278250e-02
## GO:0042476   32/2229 125/15817 4.559012e-04 3.511623e-02 3.278250e-02
## GO:0014068   22/2229  75/15817 4.761589e-04 3.620639e-02 3.380022e-02
## GO:1904064   39/2229 163/15817 5.141942e-04 3.860361e-02 3.603812e-02
## GO:0032409   61/2229 287/15817 5.581850e-04 4.112883e-02 3.839552e-02
## GO:0086003   21/2229  71/15817 5.616987e-04 4.112883e-02 3.839552e-02
## GO:0003012   83/2229 417/15817 5.925271e-04 4.285705e-02 4.000889e-02
## GO:0086011   11/2229  27/15817 6.256207e-04 4.470550e-02 4.173449e-02
## GO:0001504   15/2229  44/15817 6.637608e-04 4.641541e-02 4.333077e-02
## GO:0030490   17/2229  53/15817 6.652015e-04 4.641541e-02 4.333077e-02
## GO:0043266   27/2229 102/15817 7.062726e-04 4.870817e-02 4.547116e-02
## GO:0015844   23/2229  82/15817 7.197003e-04 4.906370e-02 4.580307e-02
##                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      geneID
## GO:0002181                                                                                         RPS7/RPS18/RPL12/RPL5/RPS2/RPL26/RPS6/RPS8/RPL22L1/RPL35/RPL7/RPL6/RPL17/EIF3E/RPS15A/EIF4B/RPL10A/RPL13/RPS17/RPL23/RPL11/RPL4/RPL13A/RPL7A/RPS3/RPLP0/PABPC1/RPL18/RPL9/RPS19/RBM24/RPL18A/RPL36/RACK1/RPL29/RPL14/RPL8/RPL41/RPS27/RPL35A/RPS25/RPS14/RPL31/RPS20/RPS24/EIF2S3/RPL38/RPL37/EIF3H/RPL27A/RPS3A/EIF3A/RPL3/RPS12/RPS9/RPL19/RPL32/RPS5/RPL34/RPS27A/RPL39/RPL30/RPL22/RPL24/RPL21/RPS23/RPL36A/RPS21/DPH5/RPL27/RPL15/RPS13/EIF3D/RPS4X/RPS16/RPS28/RPL37A/RPLP2/RPLP1/RPL28/RPS11/RPSA/IGF2BP1/DPH2/RPS15/CPEB4/RPS29/LIN28A/RPL23A/UBA52/EIF2D/EIF3M/RPS26/FAU/RPL10/CPEB2/YBX3/EIF3C
## GO:0042255                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     RPL5/NPM1/RPL6/RPL11/RPLP0/RPS19/RPS27/RPS14/NOP53/RRS1/RPL38/EIF2A/RPS5/NIP7/RPL24/RPF2/MRTO4/RPS28/RPSA/RPS15/RPL23A/BOP1/PPAN/MDN1/PRKDC/C1QBP/RPL10/PWP2
## GO:0007156                                                                                                                                                                                                                                                                                                                                             ROBO1/CNTN6/ROBO3/PCDHGC3/BSG/IGSF21/PCDHGC4/PALLD/PCDH1/PVR/PCDHB7/PCDH7/KIRREL3/AMIGO2/CDH4/PLXNB3/AMIGO1/PCDHB8/CADM4/NECTIN4/DCHS1/PCDHA2/FAT1/PCDHB6/PCDHA1/PCDH9/PCDH11Y/PCDHGA2/PCDHGB6/CDHR3/MYOT/PCDHGC5/CDHR1/FAT4/PCDHA3/PCDHGA1/CDH1/CDH12/CELSR1/CDH19/PCDHGA3/CDH23/CDH18/CDHR5/DSC3/PCDHGA9/PCDHGA7/CNTN2/DSC2/DSG1/PCDHGA8/CADM2/RET
## GO:0042273                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             RPL5/NPM1/RPL26/RPL35/RPL7/RPL6/RPL10A/RPL11/RPL7A/RPLP0/RPL14/RPL35A/NOP53/RRS1/GTPBP4/RPL38/NIP7/RPL24/RPF2/RSL1D1/MRTO4/NOP16/WDR12/MAK16/RPL23A/BOP1/GTF3A/PPAN/TMA16/MDN1/RPL10
## GO:0098742                                                                                                                                                                                                                    ROBO1/BMP2/CNTN6/ROBO3/PCDHGC3/BSG/LRRC4B/ARVCF/IGSF21/PCDHGC4/PALLD/SLITRK1/PCDH1/PVR/PCDHB7/PCDH7/SELE/KIRREL3/AMIGO2/CDH4/PLXNB3/AMIGO1/ALCAM/TENM1/PCDHB8/CPLANE2/CADM4/NECTIN4/MMP24/NTNG1/DCHS1/CEACAM19/PCDHA2/FAT1/UNC5D/PCDHB6/PCDHA1/TENM4/PCDH9/PCDH11Y/PCDHGA2/PCDHGB6/CDHR3/MYOT/CXADR/PCDHGC5/CDHR1/SLITRK2/FAT4/PCDHA3/PCDHGA1/CDH1/CDH12/CLDN1/CELSR1/CDH19/MDGA1/PCDHGA3/CDH23/CDH18/CDHR5/DSC3/PCDHGA9/PCDHGA7/CNTN2/LRRC4/DSC2/DSG1/PCDHGA8/CADM2/SELP/RET
## GO:0042274                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               RPS7/NPM1/RPS6/RPS8/RPS17/RPS19/RPS27/RPS25/RPS14/RRS1/RPS24/RPL38/RPP40/RPS5/NOB1/RPS21/RPS16/RPS28/WDR3/RPSA/NOP14/UTP20/RPS15/WDR43/BYSL/RCL1/DCAF13/PRKDC/PWP2
## GO:0000027                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              RPL5/RPL6/RPL11/RPLP0/NOP53/RRS1/RPL38/RPL24/RPF2/MRTO4/RPL23A/BOP1/PPAN/MDN1/RPL10
## GO:0042254                                                                                                                                                                                                                                                 RPS7/RPL5/NPM1/GPATCH4/RPL26/RPS6/RPS8/RPL35/RPL7/RPL6/RPL10A/RPS17/RPL11/RPL7A/RPLP0/FBL/RPS19/RPL14/DDX21/RPS27/RPL35A/RPS25/RPS14/NOP53/RRS1/LYAR/GTPBP4/RPS24/RPL38/GNL2/EIF2A/NPM3/RPP40/RPS5/NOB1/NIP7/RPL24/RPS21/RPF2/RSL1D1/RPL27/MRTO4/NOP16/RPS16/RPS28/WDR3/UTP14A/RPSA/WDR12/MAK16/NOP14/PA2G4/UTP20/RPS15/WDR43/BYSL/RPL23A/ABCE1/BOP1/GTF3A/RRP9/PPAN/RCL1/DDX10/EXOSC4/DCAF13/TMA16/MDN1/PRKDC/C1QBP/EXOSC5/RPL10/ERI1/GLUL/PWP2
## GO:0034765 CACNA1A/LRRC38/NALCN/GSG1L/FXYD6/PDE4B/KCNQ3/KCNIP1/TMC2/CABP2/CLIC4/FGF14/KCNJ5/KCNA1/KCNG1/RGN/RASGRF1/CRACR2A/PHB2/F2/HECW2/FXYD2/DPP6/NLGN3/SCN4B/CACNG7/TMC1/KCNJ9/CLCNKB/KCNK12/WNK3/SNCA/GSTO1/CACNG4/TESC/ABCC8/JPH1/HECW1/KCNA5/SLC43A1/JSRP1/KCNH7/TRPC3/KCNJ10/KCNA2/CASQ1/ANTKMT/P2RX3/CFTR/NEDD4/GPD1L/AMIGO1/KCNK10/ARC/SCN2B/LRRC26/PLP1/UBASH3B/CLCN1/KEL/SCN1B/FKBP1B/CAV3/NOS1/AGT/EDN1/KCNJ8/NTSR1/KCNK17/SHISA7/KCNJ16/HCN4/CEMIP/AKAP5/KCNIP3/CLIC1/PPP3R2/CACNA1B/KCNE5/PIRT/PDE4D/KCNJ1/APLNR/ADCYAP1R1/ATP1A2/CACNA2D2/KCNC1/KCNE3/KCNJ3/SHANK1/KCND3/BDKRB1/STOM/ADRA2A/ARG1/KCNJ12/CNKSR3/CHRM3/CNIH2/KCNJ15/CACNA1G/CACNB3/SLC26A5/DPP10/LRRC55/KCNIP4/CAV1/EDN3/MMP9
## GO:0050804                                                                                         CACNA1A/NALCN/SLC6A4/GRIN2C/ERC1/RAPSN/GRID2IP/NEURL1/NTF3/MAPT/RPL22/FRRS1L/RASGRF1/SYT11/CDC20/DISC1/ROR2/NLGN3/GFAP/CACNG7/IQSEC2/PRKCG/CAMK2A/DCC/CSPG5/SNCAIP/CD38/SNCA/CRTC1/CACNG4/SRC/SYT12/ATF4/SYT4/CHRNA5/SLC4A8/KCNJ10/TNR/GRM4/P2RX3/SCGN/STXBP1/DGKI/GRM8/GABBR1/SLC12A2/FAM107A/NRG3/ARC/NEUROD2/NTNG1/AGT/EDN1/PTN/GRIN3A/CELF4/SHISA7/GRIK3/WNT7A/ADORA1/AKAP5/PRKCB/CACNA1B/NSG1/OXTR/CYP46A1/ITPKA/SORCS2/BCHE/GRM7/ATP1A2/ACHE/ASIC1/CDH1/SORCS3/SHANK1/BAIAP2/SLC24A2/NPTX2/ADRA2A/CNR2/SLC1A1/NTRK2/GLUL/SLC7A11/CNIH2/APBA1/CNTN2/LRRC4/PHF24/TMEM108/GRM1/P2RY1/TACR1/SYT1/GRIK4
## GO:0099177                                                                                         CACNA1A/NALCN/SLC6A4/GRIN2C/ERC1/RAPSN/GRID2IP/NEURL1/NTF3/MAPT/RPL22/FRRS1L/RASGRF1/SYT11/CDC20/DISC1/ROR2/NLGN3/GFAP/CACNG7/IQSEC2/PRKCG/CAMK2A/DCC/CSPG5/SNCAIP/CD38/SNCA/CRTC1/CACNG4/SRC/SYT12/ATF4/SYT4/CHRNA5/SLC4A8/KCNJ10/TNR/GRM4/P2RX3/SCGN/STXBP1/DGKI/GRM8/GABBR1/SLC12A2/FAM107A/NRG3/ARC/NEUROD2/NTNG1/AGT/EDN1/PTN/GRIN3A/CELF4/SHISA7/GRIK3/WNT7A/ADORA1/AKAP5/PRKCB/CACNA1B/NSG1/OXTR/CYP46A1/ITPKA/SORCS2/BCHE/GRM7/ATP1A2/ACHE/ASIC1/CDH1/SORCS3/SHANK1/BAIAP2/SLC24A2/NPTX2/ADRA2A/CNR2/SLC1A1/NTRK2/GLUL/SLC7A11/CNIH2/APBA1/CNTN2/LRRC4/PHF24/TMEM108/GRM1/P2RY1/TACR1/SYT1/GRIK4
## GO:0098657                                                                                                                                                                                                                                                                          CACNA1A/SLC6A4/SLC39A4/SLC29A2/SLC1A6/KCNJ5/SLC7A2/SCNN1B/FXYD2/GFAP/GDNF/SLC9A2/KCNJ9/SLC6A12/EPRS1/WNK3/SNCA/ABCC8/SLC7A3/SLC43A1/SLC22A4/SCNN1G/KCNJ10/SLC29A1/SLC6A3/SLC9A5/SLC18A2/SLC12A2/SLC39A14/NOS1/TRPV5/LRRC8E/AGT/SLC12A8/KCNJ8/NTSR1/KCNJ16/HCN4/AKAP5/PPP3R2/CACNA1B/TRPM1/MAPK15/KCNJ1/CNGA3/ATP1A2/KCNJ3/STEAP4/GPM6B/TRPV3/SLC24A2/ARG1/KCNJ12/SLC1A1/SLC9C1/SLC7A11/KCNJ15/TFR2/GRM1/SLC12A5/SLC6A13
## GO:0061564                                                                             PAX6/ROBO1/NTN5/NTN3/KIAA0319/CHL1/RGMA/GDF7/PLXNC1/CNTN6/ROBO3/ULK2/FEZ2/MAPT/RND2/RPL24/BSG/SEMA4D/VCL/MAP6/DISC1/EPHA7/NLGN3/VASP/HSP90AB1/GDNF/NOVA2/FEZF2/DCC/PALLD/CSPG5/SLITRK1/LHX1/ISL1/KALRN/PAK3/POU3F2/NOTCH2/ATP8A2/TRIO/BARHL2/CCKAR/EPHB4/EPHB3/DLX5/NKX6-1/CDK5R1/TNR/INPP5F/MAP2/MTR/CDH4/PLXNB3/STXBP1/AMIGO1/EPHA5/ALCAM/PTPRO/LMX1A/TSPO/KIF5A/SLIT1/PLP1/KEL/SLIT3/SCN1B/FKBP1B/RHOH/TSPAN2/DCLK1/NTNG1/LAMC2/EDN1/BCL11B/PTN/UNC5D/MT3/WNT7A/EPHA2/NEFL/LHX9/FN1/MYOT/GRM7/NRCAM/PARD6B/EDN2/SLITRK2/LHX4/LGR6/NEFH/CDH1/BAIAP2/TIAM1/RTN4RL1/NTRK2/NR4A2/VASH2/VAX1/CNTN2/SMO/SEMA3E/EDN3/RET
## GO:0050808                                                                                       TPBG/SPOCK2/ERC1/RAPSN/PPFIA4/NEURL1/RAB39B/PCDHGC3/MAPT/BCAN/SEMA4D/FRRS1L/LRRC4B/GABRB3/NRXN2/CDC20/DISC1/EPHA7/IGSF21/NLGN3/GDNF/PPFIBP2/PCDHGC4/SLITRK1/ELFN1/CTTNBP2/PAK3/FZD5/SNCA/COL4A5/EPHB3/CDK5R1/TNR/NTRK3/KIRREL3/AMIGO2/CHRNA1/NEDD4/AMIGO1/LGI2/LZTS3/PTPRO/LMX1A/GPM6A/ARC/SLIT1/NEUROD2/SRCIN1/CBLN4/NTNG1/WASF1/ACTG1/SHISA7/SNCB/PPFIBP1/GLRB/WNT7A/PCDHB6/PPFIA2/C1QL3/NEFL/OXTR/MUSK/CAMKV/ITPKA/DNM3/MYOT/PRMT3/LRRTM3/C5AR1/NRCAM/PCDHGC5/ACHE/SLITRK2/FRMPD4/NEFH/CDH1/SHANK1/BAIAP2/MDGA1/TNNI3K/SLC1A1/NTRK2/TANC2/SLC7A11/COL4A1/CNKSR2/CACNB3/CNTN2/LRRC4/SEMA3E/TMEM108/C1QL1
## GO:0006364                                                                                                                                                                                                                                                                                                                                                               RPS7/RPL5/RPL26/RPS6/RPS8/RPL35/RPL7/RPL10A/RPS17/RPL11/RPL7A/FBL/RPS19/RPL14/DDX21/RPS27/RPL35A/RPS25/RPS14/NOP53/RRS1/LYAR/GTPBP4/RPS24/NPM3/RPP40/NOB1/RPS21/RPF2/RSL1D1/RPL27/MRTO4/RPS16/RPS28/WDR3/UTP14A/WDR12/MAK16/NOP14/PA2G4/UTP20/RPS15/WDR43/BYSL/BOP1/RRP9/PPAN/RCL1/DDX10/EXOSC4/DCAF13/MDN1/PRKDC/EXOSC5/ERI1/PWP2
## GO:0071805                                                                                                                                                                                                                                                                                                                                                  LRRC38/NALCN/KCNQ3/KCNIP1/KCNJ5/KCNA1/KCNG1/FXYD2/DPP6/SLC9A2/KCNJ9/KCNK12/WNK3/ABCC8/KCNA5/KCNH7/KCNJ10/KCNA2/SLC9A5/NEDD4/AMIGO1/HPN/KCNK10/SLC12A2/LRRC26/KEL/CAV3/SLC12A8/KCNJ8/KCNK17/KCNJ16/KCNT2/HCN4/KCNIP3/KCNT1/KCNE5/KCNJ1/ATP1A2/KCNC1/KCNE3/KCNJ3/KCND3/SLC24A2/KCNJ12/SLC9C1/KCNJ15/SLC24A5/DPP10/LRRC55/SLC12A5/KCNIP4/CAV1/EDN3
## GO:0097485                                                                                                                                                                                                                                                                                                                                                             PAX6/ROBO1/NTN5/NTN3/CHL1/GDF7/PLXNC1/CNTN6/ROBO3/FEZ2/RPL24/BSG/SEMA4D/DPYSL4/EPHA7/VASP/GDNF/NOVA2/FEZF2/DCC/PALLD/LHX1/ISL1/KALRN/NOTCH2/TRIO/EPHB4/EPHB3/DLX5/CDK5R1/TNR/CDH4/PLXNB3/EPHA5/ALCAM/PTPRO/LMX1A/KIF5A/SLIT1/SLIT3/SCN1B/RHOH/LAMC2/EDN1/BCL11B/UNC5D/EPHA2/LHX9/MYOT/NRCAM/LHX4/LGR6/VAX1/CNTN2/SMO/SEMA3E/EDN3/RET
## GO:0001764                                                                                                                                                                                                                                                                                                                                                                                                   PAX6/KIAA0319/CHL1/NEXMIF/ADGRG1/FGFR1/DISC1/EMX2/FEZF2/CAMK2A/DCC/LHX1/ASTN2/BARHL2/CCKAR/SPOCK1/NKX6-1/ZMIZ1/OLIG3/CDK5R1/NTRK3/KIRREL3/NAV1/GPM6A/NRG3/DDIT4/TUBB2A/DCLK1/NTNG1/UNC5D/PHACTR1/SH3RF1/ASTN1/NDNF/NRCAM/CELSR1/MDGA1/WDR62/NTRK2/NR4A2/VAX1/CXCR4/ULK4/CNTN2/DCX/SEMA3E/EOMES
## GO:0006813                                                                                                                                                                                                                                                                                                                          LRRC38/NALCN/KCNQ3/KCNIP1/KCNJ5/KCNA1/KCNG1/FXYD2/DPP6/SLC9A2/KCNJ9/KCNK12/WNK3/ATF4/ABCC8/KCNA5/KCNH7/KCNJ10/KCNA2/SLC9A5/NEDD4/AMIGO1/HPN/KCNK10/SLC12A2/LRRC26/KEL/CAV3/NOS1/SLC12A8/KCNJ8/KCNK17/KCNJ16/KCNT2/HCN4/ADORA1/KCNIP3/KCNT1/KCNE5/KCNJ1/ATP1A2/KCNC1/KCNE3/KCNJ3/KCND3/SLC24A2/ADRA2A/KCNJ12/SLC9C1/KCNJ15/SLC24A5/DPP10/LRRC55/SLC12A5/KCNIP4/CAV1/EDN3
## GO:0050878                                                                                                                                                                                    NPR3/RPLP0/NEURL1/SLC29A2/AQP5/PDGFA/AQP6/VCL/SCNN1B/APRT/P2RY12/DGKG/F2/GHRHR/FOXA2/PLSCR1/TFAP2B/SRC/SCNN1G/SYK/SLC29A1/SLC6A3/HSPB1/CFTR/STXBP1/DGKI/ABAT/PTPRO/DGKK/DGKB/UBASH3B/TRPV5/EDN1/ANXA2/MFSD2B/TFPI2/ST3GAL4/ACTG1/ADORA1/ZBTB7B/CLIC1/MPL/GRHL3/HGFAC/EMP2/OXTR/FN1/APLNR/PRSS56/NEUROG1/FGF10/TRAF3IP2/GRHL1/SFN/CDO1/ITGA2/KLKB1/CLDN1/EMILIN1/SERPING1/ADRA2A/ENTPD2/CYP4F12/SCUBE1/PAM/CHRM3/CYP4F11/SLC7A11/ADAMTS18/STAT5A/MYL9/ALOX12B/SLC4A1/DGKH/P2RY1/TACR1/SELP/CAV1/F2RL1/HK2/FZD6
## GO:0016072                                                                                                                                                                                                                                                                                                                             RPS7/SLFN13/RPL5/RPL26/RPS6/RPS8/RPL35/RPL7/RPL10A/RPS17/RPL11/RPL7A/FBL/RPS19/RPL14/DDX21/RPS27/RPL35A/RPS25/RPS14/NOP53/RRS1/LYAR/GTPBP4/RPS24/NPM3/RPP40/NOB1/MAPT/RPS21/RPF2/NCL/RSL1D1/RPL27/MRTO4/RPS16/RPS28/WDR3/UTP14A/WDR12/MAK16/NOP14/PA2G4/UTP20/RPS15/WDR43/BYSL/BOP1/GTF3A/RRP9/PPAN/RCL1/TP53/DDX10/EXOSC4/DCAF13/MDN1/PRKDC/POLR1E/EXOSC5/ERI1/PWP2
## GO:0007411                                                                                                                                                                                                                                                                                                                                                                    PAX6/ROBO1/NTN5/NTN3/CHL1/GDF7/PLXNC1/CNTN6/ROBO3/FEZ2/RPL24/BSG/SEMA4D/EPHA7/VASP/GDNF/NOVA2/FEZF2/DCC/PALLD/LHX1/ISL1/KALRN/NOTCH2/TRIO/EPHB4/EPHB3/DLX5/CDK5R1/TNR/CDH4/PLXNB3/EPHA5/ALCAM/PTPRO/LMX1A/KIF5A/SLIT1/SLIT3/SCN1B/RHOH/LAMC2/EDN1/BCL11B/UNC5D/EPHA2/LHX9/MYOT/NRCAM/LHX4/LGR6/VAX1/CNTN2/SMO/SEMA3E/EDN3/RET
## GO:0007409                                                                                                                                                  PAX6/ROBO1/NTN5/NTN3/KIAA0319/CHL1/GDF7/PLXNC1/CNTN6/ROBO3/ULK2/FEZ2/MAPT/RND2/RPL24/BSG/SEMA4D/VCL/MAP6/DISC1/EPHA7/NLGN3/VASP/HSP90AB1/GDNF/NOVA2/FEZF2/DCC/PALLD/SLITRK1/LHX1/ISL1/KALRN/PAK3/POU3F2/NOTCH2/ATP8A2/TRIO/BARHL2/CCKAR/EPHB4/EPHB3/DLX5/NKX6-1/CDK5R1/TNR/MAP2/CDH4/PLXNB3/STXBP1/AMIGO1/EPHA5/ALCAM/PTPRO/LMX1A/KIF5A/SLIT1/KEL/SLIT3/SCN1B/RHOH/DCLK1/NTNG1/LAMC2/EDN1/BCL11B/UNC5D/MT3/WNT7A/EPHA2/NEFL/LHX9/FN1/MYOT/NRCAM/PARD6B/EDN2/SLITRK2/LHX4/LGR6/NEFH/CDH1/BAIAP2/TIAM1/NTRK2/NR4A2/VAX1/CNTN2/SMO/SEMA3E/EDN3/RET
## GO:0006836                                                                                                                                                                                                                                                                                                                                                         SLC6A4/SV2B/RPH3A/SLC29A2/SLC1A6/SLC6A7/NRXN2/SYT11/GFAP/GDNF/PRKCG/CAMK2A/CSPG5/SNCAIP/SLC6A12/SYT5/CADPS2/SNCA/SYT12/SYT4/CHRNA5/SLC4A8/KCNJ10/SLC29A1/SLC6A3/SLC18A2/GRM4/STXBP1/DOC2B/RPH3AL/LIN7A/NOS1/GRIN3A/SV2A/WNT7A/PTPRN2/OTOF/PRKCB/CADPS/ATP1A2/GPM6B/ASIC1/ICA1/ADRA2A/SLC1A1/SYT2/APBA1/SLC6A8/P2RY1/SLC18A3/SLC6A13/SYT1
## GO:0043270                                                                                                                                                                                                                                                                                         LRRC38/ATP2C2/SLC6A4/KCNA1/P2RY12/RGN/CRACR2A/F2/FXYD2/NLGN3/SCN4B/GDNF/CAMK2A/WNK3/SNCA/GSTO1/CACNG4/TESC/ATF4/ABCC8/SYT4/TRPC3/CASQ1/ANTKMT/STC1/P2RX3/CFTR/STXBP1/GPD1L/AMIGO1/TNFRSF11A/ABAT/GABBR1/SLC12A2/TSPO/ARC/LRRC26/PLP1/SCN1B/NOS1/AGT/EDN1/NTSR1/CEMIP/ADORA1/AKAP5/PPP3R2/KCNE5/PIRT/OXTR/APLNR/ADCYAP1R1/NKX2-5/KCNC1/BDKRB1/TRPV3/ADRA2A/JAK3/CNKSR3/CACNB3/LRRC55/P2RY1/CAV1/EDN3/SYT1
## GO:0030900                                                                                                                                                                                      PAX6/ROBO1/DCT/BMP2/LHX5/PITX1/GDF7/KCNA1/HDAC1/ADGRG1/BCAN/P2RY12/ATIC/BMERB1/DISC1/GHRHR/EMX2/FEZF2/IGF2BP1/GLI1/LHX1/ISL1/POU3F2/CCDC85C/CCKAR/DCLK2/SRC/PITX2/EPHB3/CCDC141/DLX5/ZMIZ1/TAL2/SLC6A3/CDK5R1/TNR/KIRREL3/ATF5/INHBA/EPHA5/HTR5A/POU3F1/LMX1A/NRG3/SLIT1/DNAJB1/DCLK1/PGAP1/BCL11B/DLX2/CHD5/PHACTR1/WNT7A/NEFL/ZIC1/NDNF/OXTR/ATP1A2/KCNC1/H2AX/FGF10/FAT4/KIF14/CDH1/RAC3/MDGA1/WDR62/RTN4RL1/NTRK2/NEUROD6/SLC7A11/NR4A2/ARHGAP11B/CXCR4/CNTN2/SMO/SEMA3E/SALL1/TMEM108/EOMES/HES5/NHLH2
## GO:2000027                                                                                                                                                                                                                                                                                                                                                                                                                                                                              ROBO1/BMP2/PDGFA/DACT1/PHB2/ROR2/GDNF/RSPO2/LHX1/SOX8/WNT9B/JHY/APCDD1/SAPCD2/CAV3/AGT/EDN1/TNFRSF11B/WNT7A/GRHL3/PRICKLE1/FGF7/ROR1/ARHGEF19/CITED2/FGF10/RSPO3/TIAM1/CELSR1/FZD7/WNT10A/SPEF1/SMO/NTN4/SNAI2/FZD6
## GO:0000028                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         RPS19/RPS27/RPS14/RPL38/RPS5/RPS28/RPSA/RPS15/PRKDC/PWP2
## GO:0042391                                                                                                                               NALCN/GRIN2C/RACK1/KCNQ3/SLC1A6/KCNJ5/KCNA1/MAPT/GABRA5/SLC4A4/GABRB3/OPRD1/RGS7BP/NLGN3/SCN4B/KCNK12/SNCA/CRTC1/SRC/KCNA5/PYCR1/INSYN2A/CHRNA5/KCNH7/SLC4A8/KCNJ10/SLC29A1/KCNA2/SLC4A3/CHRNA1/P2RX3/CFTR/NEDD4/GPD1L/GABRR2/KCNK10/ABAT/GABBR1/TSPO/CHRND/SCN2B/CLCN1/SCN1B/FKBP1B/CAV3/GLRA2/EDN1/KCNJ8/GRIN3A/CELF4/NTSR1/KCNK17/GRIK3/HCN4/GLRB/WNT7A/ADORA1/GABRB1/CLIC1/KCNE5/ATP1A2/KCNC1/CXADR/NRCAM/KCNE3/KCNJ3/BVES/GABRG3/ASIC1/NTSR2/SHANK1/BAIAP2/KCND3/CNR2/NTRK2/MYOC/CNIH2/BEST2/CACNA1G/CACNB3/DSC2/SLC26A5/TMEM108/GRM1/PMAIP1/TACR1/CAV1/GRIK4
## GO:1904062                                                                                                                                                                                                 LRRC38/GSG1L/FXYD6/PDE4B/KCNIP1/TMC2/CABP2/FGF14/KCNA1/KCNG1/RGN/RASGRF1/CRACR2A/PHB2/F2/HECW2/FXYD2/DPP6/NLGN3/SCN4B/CACNG7/TMC1/WNK3/SNCA/GSTO1/CACNG4/TESC/ABCC8/JPH1/HECW1/SLC43A1/JSRP1/TRPC3/CASQ1/ANTKMT/P2RX3/NEDD4/GPD1L/AMIGO1/ARC/SCN2B/LRRC26/PLP1/UBASH3B/KEL/SCN1B/FKBP1B/CAV3/NOS1/AGT/EDN1/NTSR1/SHISA7/HCN4/CEMIP/AKAP5/KCNIP3/PPP3R2/KCNE5/PIRT/PDE4D/APLNR/ADCYAP1R1/ATP1A2/KCNC1/KCNE3/SHANK1/BDKRB1/STOM/ADRA2A/ARG1/CNKSR3/CNIH2/CACNB3/DPP10/LRRC55/KCNIP4/CAV1/EDN3/MMP9
## GO:0048880                                                                                                                                                            PAX6/PLAAT1/PBX3/PBX1/IMPDH2/AQP5/PKNOX1/CLIC4/HDAC1/RPL24/RPGRIP1/P2RY12/SALL2/LHX1/PROM1/ISL1/COL8A1/MEIS3/SOX8/NOTCH2/FZD5/ATP8A2/TULP1/BARHL2/TFAP2B/ATF4/PITX2/TGIF2/WNT9B/SLC6A3/IFT122/INHBA/TMEM231/SMARCD3/FBN2/GPM6A/SIX5/SIX6/FREM2/CRYBG3/PRPH2/SCO2/BCL11B/DLX2/CELF4/OSR2/FAT1/FZD4/FGF9/WNT7A/USP45/LRP5L/GRHL3/RCN1/EPHA2/SH3PXD2B/TMOD1/CRYGN/PRSS56/BFSP2/CITED2/FGF10/ACHE/LRP5/EFEMP1/SERPINF1/SPRED3/SLC1A1/NTRK2/SLC7A11/ADAMTS18/COL5A1/COL4A1/COL5A2/WNT16/DLL1/VAX1/DCX/SLC44A4/HES5/GNGT1/UNC45B/CPAMD8/RET
## GO:0098659                                                                                                                                                                                                                                                                                                                                                                                                                                                                  CACNA1A/SLC39A4/KCNJ5/SCNN1B/FXYD2/SLC9A2/KCNJ9/WNK3/ABCC8/SCNN1G/KCNJ10/SLC9A5/SLC12A2/SLC39A14/TRPV5/SLC12A8/KCNJ8/KCNJ16/HCN4/AKAP5/PPP3R2/CACNA1B/TRPM1/KCNJ1/CNGA3/ATP1A2/KCNJ3/TRPV3/SLC24A2/KCNJ12/SLC9C1/KCNJ15/SLC12A5
## GO:0099587                                                                                                                                                                                                                                                                                                                                                                                                                                                                  CACNA1A/SLC39A4/KCNJ5/SCNN1B/FXYD2/SLC9A2/KCNJ9/WNK3/ABCC8/SCNN1G/KCNJ10/SLC9A5/SLC12A2/SLC39A14/TRPV5/SLC12A8/KCNJ8/KCNJ16/HCN4/AKAP5/PPP3R2/CACNA1B/TRPM1/KCNJ1/CNGA3/ATP1A2/KCNJ3/TRPV3/SLC24A2/KCNJ12/SLC9C1/KCNJ15/SLC12A5
## GO:0021953                                                                                                                                                                                                                                                                                                                                                                                                                                     PAX6/LHX5/MNX1/GDF7/MAPT/FAIM2/DISC1/HSP90AB1/NOVA2/FEZF2/DCC/LHX1/ISL1/LBX1/HOXC10/DCLK2/SPOCK1/EPHB3/NKX6-1/ZMIZ1/OLIG3/MAP2/INHBA/LMX1A/SCN1B/DCLK1/SOX4/BCL11B/DLX2/CHD5/WNT7A/GABRB1/NDNF/LHX4/RAC3/MDGA1/NTRK2/NR4A2/CNTN2/SMO/SEMA3E/EOMES/HES5/NHLH2
## GO:0007589                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      NPR3/RPLP0/NEURL1/SLC29A2/AQP5/SCNN1B/APRT/GHRHR/SLC29A1/SLC6A3/EDN1/ADORA1/ZBTB7B/OXTR/NEUROG1/FGF10/TRAF3IP2/CDO1/PAM/CHRM3/STAT5A/ALOX12B/TACR1/CAV1/HK2
## GO:0001505                                                                                                                                                                                                                                                                                                                                                              SLC6A4/RPH3A/SLC29A2/SLC1A6/NRXN2/SYT11/GFAP/GDNF/PRKCG/CAMK2A/CSPG5/SNCAIP/SLC6A12/PDE1B/SYT5/CADPS2/SNCA/SYT12/SYT4/CHRNA5/SLC4A8/KCNJ10/SLC29A1/SLC6A3/SLC18A2/GRM4/STXBP1/ABAT/DOC2B/RPH3AL/LIN7A/NOS1/GRIN3A/SV2A/WNT7A/PTPRN2/OTOF/PRKCB/MAOB/BCHE/CADPS/ATP1A2/ACHE/GPM6B/ASIC1/ADRA2A/SYT2/APBA1/SLC44A4/P2RY1/SLC6A13/SYT1
## GO:0150063                                                                                                                                                                         PAX6/PLAAT1/PBX3/PBX1/IMPDH2/AQP5/PKNOX1/CLIC4/HDAC1/RPL24/RPGRIP1/P2RY12/SALL2/LHX1/PROM1/COL8A1/MEIS3/SOX8/NOTCH2/FZD5/ATP8A2/TULP1/BARHL2/TFAP2B/ATF4/PITX2/TGIF2/WNT9B/SLC6A3/IFT122/INHBA/TMEM231/SMARCD3/FBN2/GPM6A/SIX5/SIX6/FREM2/CRYBG3/PRPH2/SCO2/BCL11B/DLX2/CELF4/OSR2/FAT1/FZD4/FGF9/WNT7A/USP45/LRP5L/GRHL3/RCN1/EPHA2/SH3PXD2B/TMOD1/CRYGN/PRSS56/BFSP2/CITED2/FGF10/ACHE/LRP5/EFEMP1/SERPINF1/SPRED3/SLC1A1/NTRK2/SLC7A11/ADAMTS18/COL5A1/COL4A1/COL5A2/WNT16/DLL1/VAX1/DCX/HES5/GNGT1/UNC45B/CPAMD8/RET
## GO:1904667                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   RPS7/RPL5/RPL23/RPL11/RPS20/RPL37/RPS15/MAD2L1
## GO:0098739                                                                                                                                                                                                                                                                                                                                                                      CACNA1A/SLC39A4/SLC1A6/KCNJ5/SLC7A2/SCNN1B/FXYD2/GFAP/SLC9A2/KCNJ9/WNK3/ABCC8/SLC7A3/SLC43A1/SLC22A4/SCNN1G/KCNJ10/SLC9A5/SLC12A2/SLC39A14/TRPV5/LRRC8E/AGT/SLC12A8/KCNJ8/NTSR1/KCNJ16/HCN4/AKAP5/PPP3R2/CACNA1B/TRPM1/KCNJ1/CNGA3/ATP1A2/KCNJ3/TRPV3/SLC24A2/ARG1/KCNJ12/SLC1A1/SLC9C1/SLC7A11/KCNJ15/GRM1/SLC12A5/SLC6A13
## GO:0006936                                                                                                                                                                                                                                                   PDE4B/KCNJ5/KCNA1/SCNN1B/GAMT/TTN/SCN4B/GDNF/ENO1/CD38/ATP8A2/GSTO1/KCNA5/PROK2/DES/JSRP1/CASQ1/CHRNA1/STC1/P2RX3/GPD1L/ABAT/SULF2/CHRND/GRIP2/TNNT2/SCN2B/CLCN1/CCDC78/SCN1B/FKBP1B/CAV3/NOS1/AGT/EDN1/KCNJ8/HCN4/ADORA1/KCNE5/TNNT3/OXTR/PDE4D/TMOD1/SPHK1/MYOT/NKX2-5/ATP1A2/NEUROG1/KCNE3/KCNJ3/EDN2/ITGA2/BDKRB2/KCND3/MYH7/ADRA2A/ACTA1/TNNI3K/KCNJ12/CHRM3/CACNA1G/MYL9/ADRA1B/SLC6A8/CXCR4/ACTA2/DSC2/MYH11/MYL1/PGAM2/TACR1/CAV1/EDN3
## GO:0001654                                                                                                                                                                                PAX6/PLAAT1/PBX3/PBX1/IMPDH2/AQP5/PKNOX1/CLIC4/HDAC1/RPL24/RPGRIP1/SALL2/LHX1/PROM1/COL8A1/MEIS3/SOX8/NOTCH2/FZD5/ATP8A2/TULP1/BARHL2/TFAP2B/ATF4/PITX2/TGIF2/WNT9B/SLC6A3/IFT122/INHBA/TMEM231/SMARCD3/FBN2/GPM6A/SIX5/SIX6/FREM2/CRYBG3/PRPH2/SCO2/BCL11B/DLX2/CELF4/OSR2/FAT1/FZD4/FGF9/WNT7A/USP45/LRP5L/GRHL3/RCN1/EPHA2/SH3PXD2B/TMOD1/CRYGN/PRSS56/BFSP2/CITED2/FGF10/ACHE/LRP5/EFEMP1/SERPINF1/SPRED3/SLC1A1/NTRK2/SLC7A11/ADAMTS18/COL5A1/COL4A1/COL5A2/WNT16/DLL1/VAX1/DCX/HES5/GNGT1/UNC45B/CPAMD8/RET
## GO:0032412                                                                                                                                                                                                                                                                                                                        LRRC38/GSG1L/FXYD6/PDE4B/CABP2/FGF14/KCNA1/KCNG1/RGN/RASGRF1/CRACR2A/PHB2/HECW2/FXYD2/NLGN3/SCN4B/CACNG7/WNK3/GSTO1/CACNG4/TESC/ABCC8/JPH1/HECW1/JSRP1/CASQ1/ANTKMT/CFTR/NEDD4/GPD1L/AMIGO1/ARC/SCN2B/LRRC26/SCN1B/FKBP1B/CAV3/NOS1/EDN1/NTSR1/SHISA7/HCN4/AKAP5/PPP3R2/KCNE5/PIRT/PDE4D/ATP1A2/KCNC1/KCNE3/SHANK1/STOM/ADRA2A/CNKSR3/CHRM3/CNIH2/CACNB3/LRRC55/CAV1/MMP9
## GO:0006865                                                                                                                                                                                                                                                                                                                                                                                                                                              SLC1A6/SLC6A7/SLC7A2/GFAP/SLC6A12/SNCA/SFXN5/SYT4/SLC7A3/SLC43A1/SLC22A4/KCNJ10/STXBP1/ABAT/GABBR1/SLC12A2/LRRC8E/AGT/SV2A/OCA2/NTSR1/ADORA1/SFXN2/GRM7/ATP1A2/SLC38A5/SLC7A10/ARG1/SLC1A1/NTRK2/SLC3A1/SLC7A11/APBA1/SLC66A1L/GRM1/SLC6A13/SLC7A14
## GO:0022613                                                                                                                                          RPS7/RPL5/NPM1/GPATCH4/RPL26/RPS6/RPS8/RPL35/RPL7/RPL6/EIF3E/EIF4B/RPL10A/RPS17/RPL11/RPL13A/RPL7A/RPLP0/FBL/RPS19/RPL14/DDX21/RPS27/RPL35A/RPS25/RPS14/NOP53/RRS1/LYAR/GTPBP4/RPS24/EIF2S3/RPL38/EIF3H/EIF3A/GNL2/EIF2A/NPM3/RPP40/RPS5/NOB1/NIP7/RPL24/CELF5/RPS21/RPF2/RSL1D1/RPL27/MRTO4/EIF3D/NOP16/RPS16/RPS28/SNRPD2/WDR3/UTP14A/HSP90AB1/RPSA/WDR12/MAK16/NOP14/PA2G4/RUVBL1/UTP20/RPS15/WDR43/BYSL/RPL23A/ABCE1/BOP1/GTF3A/EIF2D/RRP9/PPAN/RCL1/EIF3M/DDX10/EXOSC4/DCAF13/TMA16/MDN1/PRKDC/C1QBP/EXOSC5/SNRPB/RPL10/CELF4/ERI1/EIF3C/GLUL/PWP2
## GO:0022898                                                                                                                                                                                                                                                                                                                   LRRC38/GSG1L/FXYD6/PDE4B/CABP2/FGF14/KCNA1/KCNG1/RGN/RASGRF1/CRACR2A/PHB2/HECW2/FXYD2/NLGN3/SCN4B/CACNG7/WNK3/SNCA/GSTO1/CACNG4/TESC/ABCC8/JPH1/HECW1/JSRP1/CASQ1/ANTKMT/CFTR/NEDD4/GPD1L/AMIGO1/ARC/SCN2B/LRRC26/SCN1B/FKBP1B/CAV3/NOS1/EDN1/NTSR1/SHISA7/HCN4/AKAP5/PPP3R2/KCNE5/PIRT/PDE4D/ATP1A2/KCNC1/KCNE3/SHANK1/STOM/ADRA2A/CNKSR3/CHRM3/CNIH2/CACNB3/LRRC55/CAV1/MMP9
## GO:0030198                                                                                                                                                                                                                                            MFAP4/LOXL4/BMP2/SPOCK2/SPINK5/COL4A6/COL9A2/COL19A1/CRTAP/PRDX4/COL13A1/NPHS1/COL11A2/COL24A1/GFAP/COL14A1/ADAMTS14/COL8A1/LRP1/FERMT1/COL4A5/NID2/CCDC80/TNR/OLFML2B/ADAMTS2/HPN/MMP21/SULF2/CTSV/POSTN/CARMIL2/MMP24/NTNG1/AGT/ANXA2/ADAMTS19/MMP25/TNFRSF11B/SERPINH1/ADAMTS7/SH3PXD2B/NDNF/MMP23B/LOXL1/ITGA8/ADAMTS9/GPM6B/TGFB1/EMILIN1/ADAMTS8/ADAMTS18/CREB3L1/COL5A1/COL4A1/COL5A2/MATN1/RXFP1/NTN4/SMOC2/MYH11/HAPLN2/CAV1/MMP9/GAS2/MYO1E
## GO:0098656                                                                                                                                                                                                                                                                              SLC1A6/CLIC4/AQP6/GABRA5/SLC25A19/SLC7A2/SLC4A4/GABRB3/SLC25A5/ABCC4/CLCN4/SLC5A12/GFAP/SLC37A1/CLCNKB/BEST1/SLC4A2/SLC7A3/SLC43A1/SLC4A8/KCNJ10/SLC4A3/CFTR/GABRR2/SLC12A2/CLCN1/SLC26A11/LRRC8E/AGT/SLC25A32/GLRA2/SLC12A8/NTSR1/GLRB/GABRB1/CLIC1/SFXN2/ATP1A2/SLC38A5/GABRG3/SLC26A4/ADAMTS8/ARG1/SLC26A9/SLC1A1/SLC3A1/SLC7A11/BEST2/SLC66A1L/SLC6A8/SLC26A5/SLC4A1/GRM1/SLC44A4/TTYH1/SLC12A5/SLC6A13/SLC13A4
## GO:0043062                                                                                                                                                                                                                                            MFAP4/LOXL4/BMP2/SPOCK2/SPINK5/COL4A6/COL9A2/COL19A1/CRTAP/PRDX4/COL13A1/NPHS1/COL11A2/COL24A1/GFAP/COL14A1/ADAMTS14/COL8A1/LRP1/FERMT1/COL4A5/NID2/CCDC80/TNR/OLFML2B/ADAMTS2/HPN/MMP21/SULF2/CTSV/POSTN/CARMIL2/MMP24/NTNG1/AGT/ANXA2/ADAMTS19/MMP25/TNFRSF11B/SERPINH1/ADAMTS7/SH3PXD2B/NDNF/MMP23B/LOXL1/ITGA8/ADAMTS9/GPM6B/TGFB1/EMILIN1/ADAMTS8/ADAMTS18/CREB3L1/COL5A1/COL4A1/COL5A2/MATN1/RXFP1/NTN4/SMOC2/MYH11/HAPLN2/CAV1/MMP9/GAS2/MYO1E
## GO:0015701                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 SLC4A4/CYB5R2/BEST1/SLC4A2/SLC4A8/SLC4A3/CFTR/SLC26A4/CA4/SLC26A9/SLC26A5/SLC4A1
## GO:0045229                                                                                                                                                                                                                                            MFAP4/LOXL4/BMP2/SPOCK2/SPINK5/COL4A6/COL9A2/COL19A1/CRTAP/PRDX4/COL13A1/NPHS1/COL11A2/COL24A1/GFAP/COL14A1/ADAMTS14/COL8A1/LRP1/FERMT1/COL4A5/NID2/CCDC80/TNR/OLFML2B/ADAMTS2/HPN/MMP21/SULF2/CTSV/POSTN/CARMIL2/MMP24/NTNG1/AGT/ANXA2/ADAMTS19/MMP25/TNFRSF11B/SERPINH1/ADAMTS7/SH3PXD2B/NDNF/MMP23B/LOXL1/ITGA8/ADAMTS9/GPM6B/TGFB1/EMILIN1/ADAMTS8/ADAMTS18/CREB3L1/COL5A1/COL4A1/COL5A2/MATN1/RXFP1/NTN4/SMOC2/MYH11/HAPLN2/CAV1/MMP9/GAS2/MYO1E
## GO:0010959                                                                                                                                                                                            LRRC38/ATP2C2/FXYD6/TF/PDE4B/KCNIP1/STC2/TMC2/CABP2/FGF14/KCNA1/KCNG1/RGN/OPRD1/CRACR2A/F2/HECW2/FXYD2/DPP6/SCN4B/CAMK2A/TMC1/WNK3/SNCA/GSTO1/BEST1/TESC/ATF4/ABCC8/JPH1/HECW1/KCNA5/JSRP1/TRPC3/CASQ1/CRACR2B/STC1/P2RX3/NEDD4/GPD1L/AMIGO1/PLPP4/TSPO/SCN2B/LRRC26/PLP1/UBASH3B/KEL/SCN1B/FKBP1B/CAV3/NOS1/AGT/NTSR1/CEMIP/ADORA1/AKAP5/KCNIP3/PPP3R2/KCNE5/PDE4D/APLNR/ADCYAP1R1/NKX2-5/ATP1A2/KCNC1/KCNE3/BDKRB1/TRPV3/STOM/ADRA2A/JAK3/CNKSR3/CACNB3/CXCR4/GRAMD2A/DPP10/LRRC55/KCNIP4/CAV1/EDN3
## GO:0021537                                                                                                                                                                                                                                                                                                                                          PAX6/ROBO1/BMP2/LHX5/KCNA1/HDAC1/ADGRG1/BCAN/P2RY12/ATIC/BMERB1/DISC1/EMX2/FEZF2/IGF2BP1/LHX1/POU3F2/CCDC85C/DCLK2/EPHB3/CCDC141/DLX5/ZMIZ1/CDK5R1/TNR/KIRREL3/ATF5/INHBA/EPHA5/HTR5A/LMX1A/NRG3/BCL11B/DLX2/PHACTR1/NEFL/ZIC1/OXTR/ATP1A2/H2AX/FAT4/KIF14/MDGA1/WDR62/RTN4RL1/NTRK2/NEUROD6/SLC7A11/ARHGAP11B/CXCR4/CNTN2/SMO/SALL1/TMEM108/EOMES/HES5
## GO:0030879                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           ROBO1/ATP2C2/RPLP0/NEURL1/SLC29A2/APRT/PHB2/GHRHR/HOXA5/BSX/SRC/SLC29A1/SLC6A3/TNFRSF11A/NRG3/CAV3/ZBTB7B/LRP5L/HOXA9/EPHA2/OXTR/TGFA/FGF10/LRP5/CDO1/ITGA2/ARG1/PAM/CYP19A1/STAT5A/RXFP1/SMO/CAV1/HK2
## GO:0048568                                                                                                                                                                     PAX6/HLX/PBX3/PBX1/MTHFD1/RPL38/RNF112/USH1G/PDGFA/COL13A1/NOTO/PDGFC/TEAD3/EN2/NDRG4/E2F8/TEAD1/TBC1D23/GDNF/HOXA5/GLI1/KRT19/LHX1/LBX1/NOTCH2/FZD5/ATP8A2/TAL1/ATF4/HOXA3/PITX2/WNT9B/TP53/DLX5/ZFPM2/IFT122/HPN/CCDC40/FBN2/PKDCC/EYA1/HMX3/NKX3-2/HOXA4/EDN1/EN1/DLX2/RPL10/OSR2/FGF9/SNAI1/HOXA7/HOXA9/GRHL3/EPHA2/ZIC1/TMIE/TBX4/APLNR/HOXB3/NKX2-5/ITGA8/NEUROG1/CITED2/FGF10/STRC/EFEMP1/RSPO3/ZFPM1/TGFB1/TEAD4/CDH23/TBX15/POU3F4/ALX3/VASH2/PCSK5/WNT16/DLL1/SMO/SALL1/SLC44A4/SOX15/EOMES/HOXA1/RBPMS2/TTPA/FZD6
## GO:0023061                                                                                                                                            SLC6A4/ADCY5/RPH3A/PCK2/TM7SF3/ABCC4/NRXN2/SYT11/GHRHR/GDNF/PRKCG/CAMK2A/CSPG5/ISL1/FOXA2/NNAT/SNCAIP/MAFA/SYT5/CADPS2/CD38/SNCA/NOS2/CCKAR/TFAP2B/SYT12/ABCC8/SYT4/GPR68/KCNA5/CHRNA5/NKX6-1/SLC4A8/SYK/KCNA2/GRM4/CFTR/INHBA/STXBP1/EPHA5/ACSL4/TNFRSF11A/ABAT/AGTR1/DOC2B/GABBR1/RPH3AL/TSPO/LIN7A/FKBP1B/CPE/AGT/SOX4/EDN1/GRIN3A/SV2A/FZD4/PFKM/WNT7A/ADORA1/PTPRN2/LRP5L/OTOF/PRKCB/MAOB/RAB11FIP1/SLC9B2/NR1H4/OXTR/ABCA1/CADPS/LRP5/MC4R/ASIC1/ICA1/ADRA2A/GLUL/SYT2/CYP19A1/PCSK5/APBA1/TFR2/SLC44A4/HCAR2/P2RY1/TACR1/EDN3/SYT1/F2RL1/VSNL1
## GO:1990573                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   KCNJ5/FXYD2/KCNJ9/WNK3/ABCC8/KCNJ10/SLC12A2/SLC12A8/KCNJ8/KCNJ16/HCN4/KCNJ1/ATP1A2/KCNJ3/KCNJ12/KCNJ15/SLC12A5
## GO:0070252                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  PDE4B/KCNJ5/SCN4B/KCNA5/STC1/GPD1L/CCDC88C/TNNT2/SCN2B/SCN1B/CAV3/KCNJ8/HCN4/ADORA1/KCNE5/EMP2/PDE4D/ATP1A2/KCNE3/KCNJ3/KCND3/MYH7/CACNA1G/ACTA2/DSC2/MYL1/CAV1
## GO:0002027                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 KCNJ5/SCN4B/ISL1/KCNA5/GPD1L/SCN2B/SCN1B/FKBP1B/CAV3/AGT/EDN1/HCN4/KCNE5/PDE4D/ADM2/KCNE3/KCNJ3/EDN2/BVES/KCND3/MYH7/TNNI3K/SLC1A1/CACNA1G/ADRA1B/DSC2/CAV1/EDN3
## GO:0048708                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       PAX6/BMP2/MAPT/F2/ROR2/GFAP/LRP1/SOX8/POU3F2/NTRK3/GPR37L1/TTBK1/PLP1/TSPAN2/MT3/ROR1/C5AR1/DLL1/VAX1/CNTN2/SMO/EOMES/HES5
## GO:0010975                                                                                                                                                                        ROBO1/KIAA0319/RGMA/PLXNC1/RIT2/ULK2/TLX2/MAPT/RND2/SEMA4D/ALKAL1/SDC2/DGKG/MAP6/CDC20/NDRG4/DISC1/ROR2/HECW2/EPHA7/TRIM67/GFAP/PQBP1/NOVA2/FEZF2/DCC/SLITRK1/PAK3/PLK5/POU3F2/CD38/ATP8A2/CRTC1/SPOCK1/CREB3L2/BCL11A/HECW1/EPHB3/CDK5R1/TNR/NTRK3/INPP5F/MAP2/ARSB/NEDD4/CDH4/PLXNB3/AMIGO1/LZTS3/PTPRO/ARC/NEU4/SLIT1/KEL/SCN1B/FKBP1B/SRCIN1/CSMD3/NTNG1/AGT/PTN/DPYSL3/MT3/WNT7A/PPFIA2/NEFL/NDNF/ROR1/ITPKA/FN1/DNM3/NRCAM/SERPINF1/CDH1/BAIAP2/ID1/TIAM1/RTN4RL1/NTRK2/TANC2/FES/KLK6/ULK4/CNTN2/SEMA3E/MINAR1/RET
## GO:0043931                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        BMP2/RFLNA/SEMA4D/THBS3/CCDC154/DCHS1/ADAMTS7/SNX10/XYLT1/PHOSPHO1/ZBTB16
## GO:0000462                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  RPS8/RPS19/RPS14/RRS1/RPP40/RPS21/RPS16/NOP14/UTP20/WDR43/BYSL/RCL1/DCAF13/PWP2
## GO:0007595                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              RPLP0/NEURL1/SLC29A2/APRT/GHRHR/SLC29A1/SLC6A3/ZBTB7B/OXTR/CDO1/PAM/STAT5A/CAV1/HK2
## GO:0140694                                                                                                                                                                                                                               RPL5/NPM1/RPL6/RPL11/RPS3/RPLP0/RPS19/RPS27/RPS14/NOP53/RRS1/RPL38/EIF2A/RCC1/RPS5/ABRAXAS1/MAPT/NIP7/RPL24/MAPK9/RPS23/RPF2/MRTO4/CDC20/RPS28/TTN/RPSA/SQLE/KRT19/NEK7/RPS15/RPL23A/BOP1/SRC/PPAN/NEB/CASQ1/MDN1/TPX2/PRKDC/AURKA/C1QBP/HSPA1B/TNNT2/CCDC78/CAV3/EDN1/CDCA8/RPL10/AURKB/ACTG1/CHEK2/CEP152/SH3PXD2B/TNNT3/MAPK15/TMOD1/C10orf90/CSRP2/NKX2-5/CENPE/BIRC5/KIF11/KIFC1/KIF23/MYH7/WDR62/DLGAP5/NEBL/ACTA1/MYBL2/CHMP4C/PWP2/PRKAA2/MYL9/MYH11/DEUP1
## GO:1901796                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     MYC/RPS7/RPL5/NPM1/RPL26/RPL23/RPL11/NOP53/RRS1/RPS20/RPL37/RPF2/RPS15/BOP1/DYRK2/TP53/DYRK3/AURKA/MUC1/SOX4/AURKB/CHD5/CHEK2/SNAI1/CD44/BDKRB2/SNAI2/PMAIP1
## GO:0007193                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            NPR3/ADCY5/P2RY12/OPRD1/GRM4/HTR5A/GRM8/GPR37L1/GABBR1/EDN1/OXER1/GRIK3/ADORA1/AKAP5/PTGDR2/GRM7/CHRM4/ADRA2A/CHRM3/GABBR2/GRM1/P2RY1
## GO:0015800                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                SLC1A6/SNCA/SYT4/KCNJ10/STXBP1/ABAT/GABBR1/SLC12A2/LRRC8E/NTSR1/ADORA1/GRM7/ATP1A2/SLC1A1/NTRK2/SLC3A1/SLC7A11/APBA1/GRM1/SLC6A13
## GO:0007157                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      IGSF21/PVR/SELE/AMIGO2/CDH4/AMIGO1/ALCAM/TENM1/NECTIN4/DCHS1/CEACAM19/TENM4/CXADR/FAT4/SELP
## GO:0051444                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       RPS7/RPL5/RPL23/RPL11/RPS20/RPL37/CDC20/RPS15/BUB1B/MAD2L1
## GO:0021534                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     SLC6A4/LHX5/PSMG1/GLI1/LHX1/ATF5/GPR37L1/SMO
## GO:0021924                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     SLC6A4/LHX5/PSMG1/GLI1/LHX1/ATF5/GPR37L1/SMO
## GO:0021930                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     SLC6A4/LHX5/PSMG1/GLI1/LHX1/ATF5/GPR37L1/SMO
## GO:0070977                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        BMP2/RFLNA/SEMA4D/THBS3/CCDC154/DCHS1/ADAMTS7/SNX10/XYLT1/PHOSPHO1/ZBTB16
## GO:0048799                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    BMP2/RFLNA/SEMA4D/THBS3/CCDC154/DCHS1/ADAMTS7/SNX10/XYLT1/PHOSPHO1/ZBTB16/RET
## GO:0042363                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             CYP27B1/CYP26A1/CYP2W1/CRABP1/CYP4F12/CYP4F11/CYP4F3
## GO:1904816                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              CCT3/CCT5/CCT4/CCT2/GNL3/CCT8/CCT6A
## GO:0042476                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             BMP2/AQP5/HDAC1/AQP6/BSG/ROGDI/EDA/RSPO2/JAG2/SRC/SLC4A2/FAM20C/PITX2/CFTR/INHBA/APCDD1/CCDC154/EDN1/BCL11B/DLX2/OSR2/TNFRSF11B/ENAM/SNX10/FGF10/WNT10A/PAM/FST/SMO/TSPEAR/EDAR/DSPP
## GO:0014068                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              PDGFA/PRR5/SEMA4D/PDGFC/FGFR1/F2/ROR2/SRC/NTRK3/NEDD4/AGT/ROR1/FN1/FLT3/TEK/CD28/NTRK2/MYOC/WNT16/SEMA3E/SELP/F2RL1
## GO:1904064                                                                                                                                                                                                                                                                                                                                                                                                                                                          LRRC38/KCNA1/RGN/CRACR2A/F2/FXYD2/NLGN3/WNK3/SNCA/GSTO1/CACNG4/TESC/ABCC8/TRPC3/CASQ1/ANTKMT/P2RX3/AMIGO1/ARC/LRRC26/PLP1/NOS1/AGT/EDN1/NTSR1/CEMIP/AKAP5/PPP3R2/KCNE5/PIRT/APLNR/ADCYAP1R1/KCNC1/BDKRB1/CNKSR3/CACNB3/LRRC55/CAV1/EDN3
## GO:0032409                                                                                                                                                                                                                                                                                                                   LRRC38/GSG1L/FXYD6/PDE4B/CABP2/FGF14/KCNA1/KCNG1/RGN/RASGRF1/CRACR2A/PHB2/HECW2/FXYD2/NLGN3/SCN4B/CACNG7/WNK3/SNCA/GSTO1/CACNG4/TESC/ABCC8/JPH1/HECW1/JSRP1/CASQ1/ANTKMT/CFTR/NEDD4/GPD1L/AMIGO1/ARC/SCN2B/LRRC26/SCN1B/FKBP1B/CAV3/NOS1/EDN1/NTSR1/SHISA7/HCN4/AKAP5/PPP3R2/KCNE5/PIRT/PDE4D/ATP1A2/KCNC1/KCNE3/SHANK1/STOM/ADRA2A/CNKSR3/CHRM3/CNIH2/CACNB3/LRRC55/CAV1/MMP9
## GO:0086003                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     PDE4B/KCNJ5/SCN4B/KCNA5/STC1/GPD1L/SCN2B/SCN1B/CAV3/KCNJ8/HCN4/ADORA1/KCNE5/PDE4D/ATP1A2/KCNE3/KCNJ3/KCND3/CACNA1G/DSC2/CAV1
## GO:0003012                                                                                                                                                                                       MYOG/PDE4B/MYOD1/KCNJ5/KCNA1/FBXO32/SCNN1B/GAMT/TTN/SCN4B/GDNF/ENO1/CD38/ATP8A2/TRIM72/GSTO1/ABCC8/KCNA5/PROK2/DES/JSRP1/TRPC3/CASQ1/INPP5F/CHRNA1/STC1/P2RX3/GPD1L/ABAT/SULF2/CHRND/GRIP2/TNNT2/SCN2B/CLCN1/CCDC78/SCN1B/FKBP1B/CAV3/NOS1/SCO2/AGT/EDN1/KCNJ8/HCN4/ADORA1/KCNE5/TNNT3/OXTR/PDE4D/TMOD1/SPHK1/MYOT/NKX2-5/ATP1A2/NEUROG1/KCNE3/KCNJ3/EDN2/ITGA2/BDKRB2/KCND3/MYH7/ADRA2A/ACTA1/TNNI3K/KCNJ12/CHRM3/MYOC/CACNA1G/MYL9/ADRA1B/SLC6A8/CXCR4/ACTA2/DSC2/MYH11/MYL1/PGAM2/P2RY1/TACR1/CAV1/EDN3
## GO:0086011                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                KCNJ5/KCNA5/KCNH7/SCN2B/KCNJ8/KCNE5/KCNE3/KCNJ3/KCND3/CACNB3/CAV1
## GO:0001504                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             SLC6A4/SLC29A2/SLC1A6/GFAP/GDNF/SLC6A12/SNCA/KCNJ10/SLC29A1/SLC6A3/SLC18A2/NOS1/ATP1A2/GPM6B/SLC6A13
## GO:0030490                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  RPS8/RPS19/RPS14/RRS1/RPP40/NOB1/RPS21/RPS16/RPS28/WDR3/NOP14/UTP20/WDR43/BYSL/RCL1/DCAF13/PWP2
## GO:0043266                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                LRRC38/KCNIP1/KCNA1/KCNG1/FXYD2/DPP6/WNK3/ATF4/ABCC8/KCNA5/NEDD4/AMIGO1/LRRC26/KEL/CAV3/NOS1/ADORA1/KCNIP3/KCNE5/KCNC1/KCNE3/ADRA2A/DPP10/LRRC55/KCNIP4/CAV1/EDN3
## GO:0015844                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               SLC6A4/SYT11/GDNF/SYT5/SNCA/SYT12/SYT4/SYK/SLC6A3/KCNA2/SLC18A2/ABAT/GABBR1/NOS1/AGT/MAOB/OXTR/MAPK15/GPM6B/ADRA2A/SYT2/P2RY1/SYT1
##            Count
## GO:0002181    98
## GO:0042255    28
## GO:0007156    53
## GO:0042273    31
## GO:0098742    72
## GO:0042274    29
## GO:0000027    15
## GO:0042254    75
## GO:0034765   109
## GO:0050804    96
## GO:0099177    96
## GO:0098657    61
## GO:0061564   104
## GO:0050808    93
## GO:0006364    56
## GO:0071805    53
## GO:0097485    58
## GO:0001764    47
## GO:0006813    57
## GO:0050878    81
## GO:0016072    62
## GO:0007411    57
## GO:0007409    92
## GO:0006836    52
## GO:0043270    65
## GO:0030900    82
## GO:2000027    36
## GO:0000028    10
## GO:0042391    88
## GO:1904062    80
## GO:0048880    84
## GO:0098659    33
## GO:0099587    33
## GO:0021953    44
## GO:0007589    25
## GO:0001505    52
## GO:0150063    82
## GO:1904667     8
## GO:0098739    47
## GO:0006936    73
## GO:0001654    81
## GO:0032412    60
## GO:0006865    37
## GO:0022613    91
## GO:0022898    61
## GO:0030198    66
## GO:0098656    58
## GO:0043062    66
## GO:0015701    12
## GO:0045229    66
## GO:0010959    81
## GO:0021537    56
## GO:0030879    34
## GO:0048568    88
## GO:0023061    90
## GO:1990573    17
## GO:0070252    27
## GO:0002027    28
## GO:0048708    23
## GO:0010975    87
## GO:0043931    11
## GO:0000462    14
## GO:0007595    14
## GO:0140694    77
## GO:1901796    28
## GO:0007193    22
## GO:0015800    20
## GO:0007157    15
## GO:0051444    10
## GO:0021534     8
## GO:0021924     8
## GO:0021930     8
## GO:0070977    11
## GO:0048799    12
## GO:0042363     7
## GO:1904816     7
## GO:0042476    32
## GO:0014068    22
## GO:1904064    39
## GO:0032409    61
## GO:0086003    21
## GO:0003012    83
## GO:0086011    11
## GO:0001504    15
## GO:0030490    17
## GO:0043266    27
## GO:0015844    23
dotplot(res_GO)

10 Pathway-based analysis with rROMA

Large-scale molecular data, such as transcriptomics and proteomics, offers great opportunities for understanding the complexity of biological processes. One important aspect of data analysis in systems biology is the shift from a reductionist approach that focuses on individual components to a more integrative perspective that considers the system as a whole, where the emphasis shifted from differential expression of individual genes to determining the activity of gene sets. The rROMA algorithm is used for computation of the activity of gene sets with coordinated expression.

rRoma requires a gene expression matrix, with column names indicating samples and row names indicating gene names.

It also requires a module file containing information on the genesets that need to be evaluated. The module file can be loaded from a GMT file.

The figure below illustrates a schematic diagram of the workflow of the rROMA algorithm.

10.1 Input dataset

Warning: rRoma works with normalized data. Here the normalized TB dataset is used.

summary(log.rna.norm)
##      TB236            TB240            TB242            TB243       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 3.014   1st Qu.: 3.361   1st Qu.: 2.743   1st Qu.: 3.064  
##  Median : 5.599   Median : 5.695   Median : 5.547   Median : 5.599  
##  Mean   : 4.910   Mean   : 4.996   Mean   : 4.801   Mean   : 4.895  
##  3rd Qu.: 6.865   3rd Qu.: 6.825   3rd Qu.: 6.875   3rd Qu.: 6.840  
##  Max.   :11.559   Max.   :12.274   Max.   :12.502   Max.   :11.694  
##      TB244            TB248            TB249            TB252       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 2.937   1st Qu.: 2.943   1st Qu.: 3.106   1st Qu.: 2.777  
##  Median : 5.541   Median : 5.652   Median : 5.640   Median : 5.614  
##  Mean   : 4.872   Mean   : 4.927   Mean   : 4.903   Mean   : 4.842  
##  3rd Qu.: 6.864   3rd Qu.: 6.932   3rd Qu.: 6.868   3rd Qu.: 6.889  
##  Max.   :11.480   Max.   :12.054   Max.   :11.423   Max.   :12.332  
##      TB254            TB256            TB262            TB263       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 2.951   1st Qu.: 3.352   1st Qu.: 3.037   1st Qu.: 3.038  
##  Median : 5.609   Median : 5.701   Median : 5.659   Median : 5.562  
##  Mean   : 4.882   Mean   : 4.974   Mean   : 4.890   Mean   : 4.883  
##  3rd Qu.: 6.879   3rd Qu.: 6.863   3rd Qu.: 6.845   3rd Qu.: 6.828  
##  Max.   :11.854   Max.   :11.468   Max.   :11.405   Max.   :11.127  
##      TB264            TB267            TB268            TB269       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 3.297   1st Qu.: 3.269   1st Qu.: 3.460   1st Qu.: 2.836  
##  Median : 5.694   Median : 5.627   Median : 5.605   Median : 5.591  
##  Mean   : 4.971   Mean   : 4.956   Mean   : 4.994   Mean   : 4.858  
##  3rd Qu.: 6.831   3rd Qu.: 6.820   3rd Qu.: 6.810   3rd Qu.: 6.896  
##  Max.   :11.434   Max.   :11.141   Max.   :11.292   Max.   :11.854  
##      TB271            TB274            TB277            TB279       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 3.060   1st Qu.: 3.041   1st Qu.: 3.039   1st Qu.: 3.070  
##  Median : 5.638   Median : 5.640   Median : 5.556   Median : 5.700  
##  Mean   : 4.903   Mean   : 4.901   Mean   : 4.886   Mean   : 4.929  
##  3rd Qu.: 6.832   3rd Qu.: 6.844   3rd Qu.: 6.838   3rd Qu.: 6.869  
##  Max.   :11.615   Max.   :12.933   Max.   :11.408   Max.   :12.296  
##      TB280            TB284            TB288            TB290       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 2.945   1st Qu.: 3.061   1st Qu.: 2.828   1st Qu.: 2.574  
##  Median : 5.544   Median : 5.682   Median : 5.544   Median : 5.522  
##  Mean   : 4.833   Mean   : 4.931   Mean   : 4.817   Mean   : 4.768  
##  3rd Qu.: 6.791   3rd Qu.: 6.863   3rd Qu.: 6.867   3rd Qu.: 6.866  
##  Max.   :11.671   Max.   :12.284   Max.   :10.915   Max.   :11.612  
##      TB292            TB295            TB296            TB299       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 3.124   1st Qu.: 3.304   1st Qu.: 2.689   1st Qu.: 3.134  
##  Median : 5.588   Median : 5.611   Median : 5.644   Median : 5.658  
##  Mean   : 4.920   Mean   : 4.974   Mean   : 4.816   Mean   : 4.931  
##  3rd Qu.: 6.854   3rd Qu.: 6.845   3rd Qu.: 6.855   3rd Qu.: 6.854  
##  Max.   :11.461   Max.   :11.090   Max.   :11.815   Max.   :12.186  
##      TB300            TB303            TB305            TB081       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 3.177   1st Qu.: 3.277   1st Qu.: 3.290   1st Qu.: 3.176  
##  Median : 5.617   Median : 5.619   Median : 5.724   Median : 5.627  
##  Mean   : 4.928   Mean   : 4.967   Mean   : 4.997   Mean   : 4.931  
##  3rd Qu.: 6.817   3rd Qu.: 6.844   3rd Qu.: 6.855   3rd Qu.: 6.851  
##  Max.   :11.030   Max.   :11.493   Max.   :11.300   Max.   :11.067  
##      TB084            TB085            TB087            TB102       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 2.939   1st Qu.: 2.813   1st Qu.: 3.222   1st Qu.: 2.741  
##  Median : 5.720   Median : 5.555   Median : 5.700   Median : 5.595  
##  Mean   : 4.902   Mean   : 4.854   Mean   : 4.968   Mean   : 4.819  
##  3rd Qu.: 6.872   3rd Qu.: 6.899   3rd Qu.: 6.825   3rd Qu.: 6.865  
##  Max.   :12.914   Max.   :12.369   Max.   :12.296   Max.   :11.136  
##      TB103            TB114            TB118            TB121       
##  Min.   : 0.000   Min.   : 0.000   Min.   : 0.000   Min.   : 0.000  
##  1st Qu.: 3.148   1st Qu.: 2.927   1st Qu.: 2.525   1st Qu.: 3.137  
##  Median : 5.652   Median : 5.655   Median : 5.609   Median : 5.622  
##  Mean   : 4.914   Mean   : 4.893   Mean   : 4.801   Mean   : 4.902  
##  3rd Qu.: 6.817   3rd Qu.: 6.855   3rd Qu.: 6.952   3rd Qu.: 6.827  
##  Max.   :11.669   Max.   :12.868   Max.   :13.090   Max.   :11.489  
##      TB137       
##  Min.   : 0.000  
##  1st Qu.: 3.400  
##  Median : 5.734  
##  Mean   : 5.009  
##  3rd Qu.: 6.810  
##  Max.   :13.147

We can then load sample labels:

Group <- design$Group
names(Group) <- rownames(design)
  
table(Group)
## Group
## G1 G2 G3 G4 
##  4 11 13 13

10.2 Selecting the module list

Here, we extract all the “HALLMARK” gene sets from MSigDB (Liberzon A et al, Cell Syst 2015). The Molecular Signatures Database (MSigDB) is one of the most widely used and comprehensive databases of gene sets for performing gene set enrichment analysis. The collection of “Hallmark” gene sets as part of MSigDB consists of “refined” gene sets describing biological states or processes.

Note that we chose the Hallmark database for explanatory purpose. To have a more complete view of biological systems at stake in your study, we recommend using rRoma with multiple databases.

Hallmarks <- SelectFromMSIGdb("HALLMARK")
## [1] "Searching in MsigDB v6.2"
## [1] "The following genesets have been selected:"
##  [1] "HALLMARK_TNFA_SIGNALING_VIA_NFKB (200 genes)"          
##  [2] "HALLMARK_HYPOXIA (200 genes)"                          
##  [3] "HALLMARK_CHOLESTEROL_HOMEOSTASIS (74 genes)"           
##  [4] "HALLMARK_MITOTIC_SPINDLE (200 genes)"                  
##  [5] "HALLMARK_WNT_BETA_CATENIN_SIGNALING (42 genes)"        
##  [6] "HALLMARK_TGF_BETA_SIGNALING (54 genes)"                
##  [7] "HALLMARK_IL6_JAK_STAT3_SIGNALING (87 genes)"           
##  [8] "HALLMARK_DNA_REPAIR (150 genes)"                       
##  [9] "HALLMARK_G2M_CHECKPOINT (200 genes)"                   
## [10] "HALLMARK_APOPTOSIS (161 genes)"                        
## [11] "HALLMARK_NOTCH_SIGNALING (32 genes)"                   
## [12] "HALLMARK_ADIPOGENESIS (200 genes)"                     
## [13] "HALLMARK_ESTROGEN_RESPONSE_EARLY (200 genes)"          
## [14] "HALLMARK_ESTROGEN_RESPONSE_LATE (200 genes)"           
## [15] "HALLMARK_ANDROGEN_RESPONSE (101 genes)"                
## [16] "HALLMARK_MYOGENESIS (200 genes)"                       
## [17] "HALLMARK_PROTEIN_SECRETION (96 genes)"                 
## [18] "HALLMARK_INTERFERON_ALPHA_RESPONSE (97 genes)"         
## [19] "HALLMARK_INTERFERON_GAMMA_RESPONSE (200 genes)"        
## [20] "HALLMARK_APICAL_JUNCTION (200 genes)"                  
## [21] "HALLMARK_APICAL_SURFACE (44 genes)"                    
## [22] "HALLMARK_HEDGEHOG_SIGNALING (36 genes)"                
## [23] "HALLMARK_COMPLEMENT (200 genes)"                       
## [24] "HALLMARK_UNFOLDED_PROTEIN_RESPONSE (113 genes)"        
## [25] "HALLMARK_PI3K_AKT_MTOR_SIGNALING (105 genes)"          
## [26] "HALLMARK_MTORC1_SIGNALING (200 genes)"                 
## [27] "HALLMARK_E2F_TARGETS (200 genes)"                      
## [28] "HALLMARK_MYC_TARGETS_V1 (200 genes)"                   
## [29] "HALLMARK_MYC_TARGETS_V2 (58 genes)"                    
## [30] "HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION (200 genes)"
## [31] "HALLMARK_INFLAMMATORY_RESPONSE (200 genes)"            
## [32] "HALLMARK_XENOBIOTIC_METABOLISM (200 genes)"            
## [33] "HALLMARK_FATTY_ACID_METABOLISM (158 genes)"            
## [34] "HALLMARK_OXIDATIVE_PHOSPHORYLATION (200 genes)"        
## [35] "HALLMARK_GLYCOLYSIS (200 genes)"                       
## [36] "HALLMARK_REACTIVE_OXIGEN_SPECIES_PATHWAY (49 genes)"   
## [37] "HALLMARK_P53_PATHWAY (200 genes)"                      
## [38] "HALLMARK_UV_RESPONSE_UP (158 genes)"                   
## [39] "HALLMARK_UV_RESPONSE_DN (144 genes)"                   
## [40] "HALLMARK_ANGIOGENESIS (36 genes)"                      
## [41] "HALLMARK_HEME_METABOLISM (200 genes)"                  
## [42] "HALLMARK_COAGULATION (138 genes)"                      
## [43] "HALLMARK_IL2_STAT5_SIGNALING (200 genes)"              
## [44] "HALLMARK_BILE_ACID_METABOLISM (112 genes)"             
## [45] "HALLMARK_PEROXISOME (104 genes)"                       
## [46] "HALLMARK_ALLOGRAFT_REJECTION (200 genes)"              
## [47] "HALLMARK_SPERMATOGENESIS (135 genes)"                  
## [48] "HALLMARK_KRAS_SIGNALING_UP (200 genes)"                
## [49] "HALLMARK_KRAS_SIGNALING_DN (200 genes)"                
## [50] "HALLMARK_PANCREAS_BETA_CELLS (40 genes)"
Hallmarks <- lapply(Hallmarks, function(x){
  x$Name <- sub("HALLMARK_", "", x$Name)
  x
})

In case we already have a GMT file with modules we want to test, we can also load them by using the ReadGMTFile function.

#myDB <- ReadGMTFile("h.all.v6.1.symbols.gmt", SearchString = NULL, Mode = "ANY")

10.3 Performing rROMA: basics

We can now run rRoma on our dataset by simply specifying the expression dataset and the modules you want to test. Here we are fixing the seed for used for random permutations.

# set.seed(69)
rRoma.output <- rRoma.R(log.rna.norm, Hallmarks)

10.4 Reading the output

The most important information can be found in the module matrix, here in rRoma.output$ModuleMatrix. It contains p and q values for overdispersion (L1) and shift (Median Exp) for all tested modules.

head(rRoma.output$ModuleMatrix)
##                                   L1 Median L1 ppv L1     L1/L2 Median L1/L2
## TNFA_SIGNALING_VIA_NFKB    0.1321127 0.1553025   0.96 0.9991029     1.248812
## HYPOXIA                    0.1538242 0.1553025   0.54 1.0983751     1.248812
## CHOLESTEROL_HOMEOSTASIS    0.2230479 0.1870367   0.14 1.6991443     1.325560
## MITOTIC_SPINDLE            0.2243646 0.1553025   0.00 1.6896764     1.248812
## WNT_BETA_CATENIN_SIGNALING 0.3488909 0.2260947   0.02 2.7078359     1.459496
## TGF_BETA_SIGNALING         0.2802496 0.1982851   0.05 1.7808915     1.334400
##                            ppv L1/L2  Median Exp ppv Median Exp       q L1
## TNFA_SIGNALING_VIA_NFKB         1.00 -1.22608089           0.00 0.98979592
## HYPOXIA                         0.88 -0.73655754           0.00 0.72500000
## CHOLESTEROL_HOMEOSTASIS         0.11  0.58965308           0.00 0.33333333
## MITOTIC_SPINDLE                 0.02  0.11061512           0.46 0.00000000
## WNT_BETA_CATENIN_SIGNALING      0.00  0.09990226           0.70 0.07142857
## TGF_BETA_SIGNALING              0.11 -0.33013768           0.11 0.13888889
##                              q L1/L2 q Median Exp
## TNFA_SIGNALING_VIA_NFKB    1.0000000   0.00000000
## HYPOXIA                    1.0000000   0.00000000
## CHOLESTEROL_HOMEOSTASIS    0.3666667   0.00000000
## MITOTIC_SPINDLE            0.1111111   0.68888235
## WNT_BETA_CATENIN_SIGNALING 0.0000000   0.70480392
## TGF_BETA_SIGNALING         0.3666667   0.04941176

We are interested in two different types of modules:

- Shifted modules, whose genes behave differently from the rest of the genes in at least one sample. Corresponding p value is in ppv Median Exp. A q value is also calculated and given in q Median Exp. Here we will consider a module as shifted if p < 0.05. We don’t look at q values here as both the number of samples and the number of tested modules is small. Consider looking at q values for larger data sets.

- Overdispersed modules, for which the approximation to one PC is correct. Corresponding p value is in ppv ML1. A q value is also calculated and given in q L1. Here we will consider a module as overdispersed if p < 0.05. We don’t look at q values for the same reason as before, but considered looking at it for larger datasets

10.4.1 Shifted modules

We are first interested in shifted modules.

shifted.modules <- which(rRoma.output$ModuleMatrix[, "ppv Median Exp"] <= 0.05)

We want to see which samples are responsible for the shift. This can be done by looking at Sample Scores. The function plots a heatmap of these score:

Plot.Genesets.Samples(rRoma.output, Selected = shifted.modules, GroupInfo = Group, cluster_cols = TRUE)
## [1] "30 genesets selected"
## [1] "41 samples have an associated group"

This representation can help us define groups of samples that behave similarly on a pathway level.

10.4.2 Overdispersed modules

overdispersed.modules <- which(rRoma.output$ModuleMatrix[, "ppv L1"] <= 0.05 & rRoma.output$ModuleMatrix[, "ppv Median Exp"] > 0.05)

We first plot the same heatmap as before to visualize sample scores:

Plot.Genesets.Samples(rRoma.output, Selected = overdispersed.modules, GroupInfo = Group, cluster_cols = TRUE)
## [1] "8 genesets selected"
## [1] "41 samples have an associated group"